New antibacterial compounds

ABSTRACT

The present invention relates to novel antibacterial compounds, pharmaceutical compositions containing them and their use as antimicrobials.

FIELD OF THE INVENTION

The present invention relates to novel antibacterial compounds,pharmaceutical compositions containing them and their use asantimicrobials.

BACKGROUND OF THE INVENTION

DNA topoisomerases are enzymes involved in the modification of theDNA-supercoiling during replication or transcription. These enzymes bindto single-stranded or double-stranded DNA and they cut the phosphatebackbone of the DNA such that the DNA strand is untangled or unwound. Atthe end of the replication or transcription processes, the enzymesthemselves reseal the DNA backbone.

DNA topoisomerases are classified as type I when they cut a singlestrand of a DNA double helix and as type II when they cut both strandsof a DNA double helix.

Bacterial type II topoisomerases comprise DNA gyrase and topoisomeraseIV (TopoIV), which are heterotetrameric enzymes concurrently present inalmost all the prokaryotic cells. Both the enzymes are necessary for DNAreplication and, hence, for bacterial cell growth and division.

Bacterial type II topoisomerases are proven antibacterial targets, inparticular of compounds belonging to fluoroquinolone class.

Fluoroquinolones are broad-spectrum antibacterial drugs that play animportant role in treatment of bacterial infections, especiallyhospital-acquired infections and infections in which resistance to otherclasses of antibacterial drugs is suspected. Fluoroquinolones act byinhibiting the DNA gyrase in Gram negative bacteria and thetopoisomerase IV in Gram positive bacteria.

However, resistance to fluoroquinolones emerged in recent years due tomutations that altered either the active site of the drug targets DNAgyrase and topoisomerase IV or the drug accumulation. In addition,resistance to quinolones can be mediated by plasmids that produce theQnr protein, which protects the quinolone targets from inhibition (G. A.Jacoby, CID, 2005:41, Suppl. 2, SD120-S126).

According to the World Health Organization, the antimicrobial resistance(AMR) is the resistance of a microorganism to an antimicrobial drug towhich it was originally sensitive. Resistant bacteria are able towithstand attack by antibiotics and antibacterial drugs, so thatstandard treatments become ineffective and infections persist increasingrisk of spread to others.

Mitton-Fry M. J. et al. (Bioorg. Med. Chem. Lett., 23, 2010, 2955-2961)developed novel quinolone derivatives as inhibitors of bacterial DNAgyrase and topoisomerase IV. Given the importance of stepwise targetmutations in the clinical history of fluoroquinolones resistance, theauthors felt strongly that providing inhibition of TopoIV alongside DNAgyrase was critically important. According to the authors, suchdual-targeting activity should slow the rate of resistance emergence inthe clinic, since organism which mutate DNA gyrase to avoid inhibitionwould still be susceptible to killing via TopoIV inhibition.

Surivet J. P. et al. (J. Med. Chem. 2013, 56, 7396-7415) reported thedesign of novel bacterial dual DNA gyrase and TopoIV inhibitorscomprising a tetrahydropyran core and demonstrated that dual inhibitionof DNA gyrase and TopoIV is required to minimize the rate of resistancedevelopment.

Zayane Marwa et al. (Journal of Enzyme Inhibition and MedicinalChemistry 2016, 31(6):1566-1575) reported the design and synthesis of4-methylumbelliferone derivatives with antimicrobial, anticoagulant, andanticholinesterase activity.

WO 2006/105289 relates to heterocyclic compounds, more particularlypyrazole compounds, which were tested for inhibition of both DNA gyraseand topoisomerase IV.

WO 02/072572, WO 2006/021448, WO 2008/139288, WO 2009/141398, WO2010/081874, WO 2010/084152, WO 2013/068948, WO 2013/080156, WO2016/027249, WO 2016/096631 and WO 2016/096686 disclose heterocycliccompounds endowed with antimicrobial activity.

WO 96/10568 and WO 2012/003418 disclose heterocyclic compounds endowedwith other therapeutic activity.

SUMMARY OF THE INVENTION

The Applicant recognized that there is a strong and continuous need forantibacterial drugs that overcome the problem of resistant bacteria.

The Applicant faced the problem to develop new antibacterial compoundsthat allow to overcome the problem of antibacterial resistance.

More in particular, the Applicant faced the problem to develop newantibacterial compounds capable of concurrently inhibit bacterial typeII topoisomerases, i.e. DNA gyrase and topoisomerase IV.

Also, the Applicant faced the problem to develop new antibacterialcompounds having broad spectrum of activity, i.e. useful against Grampositive and/or Gram negative bacteria.

Thus, in a first embodiment, the present invention relates to a compoundof formula (1):

A-L₁-Y-L₂-R—B  (1)

wherein

A is a cyclic group having one of the following formulae (I), (II) and(III):

wherein

-   -   G₁ is CH or N;    -   G₂ is CR₁ or N;    -   G₃ is CH or N when the dashed line represents a double bond, or        CH₂, NH or O when the dashed line represents a single bond;    -   G₄ is CH or N when the dashed line represents a double bond, or        CH₂, NH or O when the dashed line represents a single bond;    -   C₁ represents the atoms necessary to form an aliphatic or        aromatic six-membered cycle optionally comprising one or more        heteroatom selected from nitrogen atom and oxygen atom, said        cycle being optionally substituted by one or more substituent        selected from the group consisting of halogen atom, (C₁₋₃)alkyl        group, cyano group, oxo group (═O), and (C₁₋₃)alkoxy group; R₁        is hydrogen atom, halogen atom, cyano or (C₁₋₃)alkoxy group;

L₁ is σ bond, or (C₁₋₃)alkylenyl group, optionally substituted withhydroxy group;

Y is a ring selected from the group consisting of piperidinyl,piperazinyl, pyrrolidinyl, 1-3 cyclobutyl, 1-3 cyclopentyl, 1-2cyclopropyl, azetidinyl, azabicyclo-octyl, morpholinyl and cyclohexylring, said ring being optionally substituted by one or more substituentselected from the group consisting of hydroxy group, (C₁₋₃)alkylenyl-OHgroup, (C₁₋₃)alkylenyl-O—(C₁₋₃)alkyl group, (C₁₋₃)alkylenyl-CONR′R″group, and CONR′R″ group, wherein R′ and R″ are hydrogen atom or(C₁₋₃)alkyl;

L₂ is σ bond, —(C₁₋₃)alkylenyl group, NR′″ group, NR′″—(C₁₋₃)alkylenylgroup, (C₁₋₃)alkylenyl-NR′″ group, NR′″—(C₁₋₃)alkylenyl-NR′″ group, or(C₁₋₃)alkylenyl-NR′″—(C₁₋₃)alkylenyl group, said group being optionallysubstituted with a hydroxy group, wherein R′″ is hydrogen, (C₁₋₃)alkyl,(C₁₋₃)alkylenyl-OH, (C₁₋₃)alkylenyl-O—(C₁₋₃)alkyl, or(C₁₋₃)alkylenyl-CONR′R″, wherein R′ and R″ are hydrogen atom or(C₁₋₃)alkyl;

R is σ bond or heterocyclic ring, aliphatic or aromatic, having 5members containing one or more nitrogen atoms, optionally substitutedwith CH₂OH, CH₂CN, CN or CONR′R″, wherein R′ and R″ are hydrogen atom or(C₁₋₃)alkyl; and

B is a cyclic group having one of the following formula (IV) to (IX):

wherein

-   -   G₅ is CH or N;    -   G₆ is CH or N;    -   G₇ is CH or N;    -   G₈ is O or NR₆;    -   R₂ is hydrogen atom or halogen atom;    -   R₃ is hydrogen atom or cyano group;    -   R₄ and R₅ are hydrogen atoms, or together form a six-membered        aliphatic ring containing one oxygen atom;    -   R₆ is a hydrogen atom or a (C₁₋₃)alkyl group;    -   C₂ represents the atoms necessary to form a six-membered        aliphatic or aromatic heterocycle comprising an oxygen atom or a        nitrogen atom;    -   C₃ represents the atoms necessary to form a six-membered        aliphatic heterocycle comprising one oxygen atom and optionally        one nitrogen atom, said heterocycle being optionally substituted        by one or more substituent selected from the group consisting of        (C₁₋₃)haloalkyl group, amido group (—CONH₂), and oxo group (═O);        and    -   C₄ represents the atoms necessary to form a ten-membered fused        bicycle comprising at least one nitrogen atom and at least one        other heteroatom selected from oxygen atom and sulfur atom, said        bicycle being optionally substituted by one or more substituent        selected from the group consisting of halogen atom, (C₁₋₃)alkyl        group, cyano group, and oxo group (═O);

with the proviso that when Y is piperidinyl ring or piperazinyl ring, ifA is represented by formula (I) and B is represented by formula (V) or(VI), then G₂ is only N,

and addition salts with pharmaceutically acceptable organic or inorganicacids or basis, enantiomers, N-oxides and quaternary ammonium salts ofsaid compound of formula (1).

In a second embodiment, the present invention relates to apharmaceutical composition comprising at least one compound of formula(1).

In a third embodiment, the present invention relates to the compounds offormula (1) for use in medicine.

In a fourth embodiment, the present invention relates to the compoundsof formula (1) for use in the treatment of bacterial infections.

In a fifth embodiment, the present invention relates to a method fortreating a bacterial infection, comprising the administration of acompound of formula (1) to a patient in need thereof.

According to a preferred aspect of the present invention, G₁ is N.

According to a preferred aspect of the present invention G₂ is CR1.

According to a preferred aspect of the present invention G₃ is CH or Nwhen the dashed line represents a double bond, or O when the dashed linerepresents a single bond.

According to a preferred aspect of the present invention G₄ is N whenthe dashed line represents a double bond, or O when the dashed linerepresents a single bond.

Preferably, R₁ is a halogen atom, a cyano group, or an alkoxy grouphaving from 1 to 2 carbon atoms.

Advantageously, R₁ is selected from the group consisting of a fluorineatom, a chlorine atom, a cyano group and a methoxy group.

Preferably, R₂ is a hydrogen atom, a fluorine atom, a chlorine atom, anda bromine atom.

Advantageously, R₂ is selected from the group consisting of a hydrogenatom and a fluorine atom.

According to a preferred embodiment, R₃ is hydrogen atom or cyano group,provided that, when R₃ is cyano group, A is different from formula(III).

Preferably, R₄ and R₅ are hydrogen atoms.

Preferably, C₁ is optionally substituted by one or more substituentselected from the group consisting of fluorine or chlorine atom, methylor ethyl group, methoxy or propoxy group, and oxo group (═O).

Advantageously, L₁ is a σ bond or a methylene group (—CH₂—).

Preferably, L₂ is a σ bond, a (C₁₋₃)alkylenyl group, NR′″ group,—NR′″—(C₁₋₃)alkylenyl group, (C₁₋₃)alkylenyl-NR′″— group,—NR′″—(C₁₋₃)alkylenyl-NR′″— group, or(C₁₋₃)alkylenyl-NR′″—(C₁₋₃)alkylenyl group, said group being optionallysubstituted with one or more hydroxy group.

More preferably, L₂ is a σ bond, a (C₁₋₂)alkylenyl group, NR′″ group,—NR′″—(C₁₋₂)alkylenyl group, (C₁₋₂)alkylenyl-NR′″— group, —NR′″—(C₁₋₂)alkylenyl-NR′″— group, or (C₁₋₂)alkylenyl-NR′″—(C₁₋₂)alkylenyl group,said group being optionally substituted with one hydroxy group.

Preferably, R′″ is hydrogen, (C₁₋₂)alkyl, (C₁₋₂)alkylenyl-OH,(C₁₋₂)alkylenyl-O—(C₁₋₂)alkyl, or (C₁₋₂)alkylenyl-CONR′R″, wherein R′and R″ are hydrogen atom or (C₁₋₂)alkyl.

Preferably, R′″ is hydrogen, methyl, CH₂—OH, or CH₂CONR′R″, wherein R′and R″ are hydrogen atom or methyl.

Preferably, R is a σ bond or an aromatic heterocyclic ring having 5members containing one or more nitrogen atoms, optionally substitutedwith CH₂CN or CN.

Advantageously, R is a σ bond, a 1H-imidazol-4-yl group, or a1H-pyrrol-2-yl group, optionally substituted with CH₂CN or CN.

Preferably, C₃ is optionally substituted by one or more substituentselected from the group consisting of (C₁₋₃)fluoroalkyl group, amidogroup (—CONH₂), or oxo group (═O). More preferably, said(C₁₋₃)fluoroalkyl group is a trifluoromethyl group.

Preferably, C₄ is optionally substituted by one or more substituentselected from the group consisting of fluoride atom, methyl group, cyanogroup, or oxo group (═O).

According to a preferred aspect of the present invention, A is a cyclicgroup having one of the following formulas:

According to a preferred aspect of the present invention, Y is a ringhaving one of the following formulae:

According to a preferred aspect of the present invention, B is a cyclicgroup having one of the following formulae:

In the present description and in the following claims, the term“(C₁₋₆)alkyl” means a linear or branched alkyl chain comprising from 1to 6 carbon atoms, such as for example methyl, ethyl, propyl, isopropyl,n-butyl, isobutyl, sec-butyl, tert-butyl, pentyl, isopentyl, sec-pentyl,tert-pentyl, neo-pentyl, 3-pentyl, hexyl, isohexyl.

In the present description and in the following claims, the term“(C₁₋₃)alkyl” means a linear or branched alkyl chain comprising from 1to 3 carbon atoms, such as for example methyl, ethyl, propyl, isopropyl.

In the present description and in the following claims, the term“(C₁₋₆)alkylenyl” means a divalent linear or branched alkyl chaincomprising from 1 to 6 carbon atoms, such as for example methylenyl(—CH₂—), ethylenyl (—CH₂CH₂—), propylenyl (—CH₂CH₂CH₂—) or butylenyl(—CH₂CH₂CH₂CH₂—).

In the present description and in the following claims, the term“(C₄₋₅)cycloalkylenyl” means a divalent cycloalkyl group comprising 4 or5 carbon atoms, such as cyclobutylenyl and cyclopentylenyl.

In the present description and in the following claims, the term“(C₁₋₃)alkoxy” means a linear or branched alkoxy chain comprising from 1to 3 carbon atoms, such as for example methoxy, ethoxy, propoxy,isopropoxy.

In the present description and in the following claims, the term“(C₁₋₃)haloalkyl” means a linear or branched haloalkyl chain comprisingfrom 1 to 3 carbon atoms wherein one or more hydrogen atoms aresubstituted by a halide atom, such as for example, fluoromethyl,chloromethyl, trifluoromethyl, trichloromethyl, fluoroethyl, chloroethylpentafluoroethyl, pentachloroethyl, fluoropropyl, chloropropyl,esafluoropropyl, and esachloroisopropyl.

Certain compounds of this invention may exist in tautomeric forms, andthis invention includes all such tautomeric forms of those compoundsunless otherwise specified.

Unless otherwise stated, structures depicted herein are also meant toinclude all stereochemical forms of the structure; i.e., the R and Sconfigurations for each asymmetric center. Thus, single stereochemicalisomers as well as enantiomeric and diastereomeric mixtures of thepresent compounds are within the scope of the invention. Thus, thisinvention encompasses each diastereomer or enantiomer substantially freeof other isomers (>90%, and preferably >95%, free from otherstereoisomers on a molar basis) as well as a mixture of such isomers.

Particular optical isomers can be obtained by resolution of the racemicmixtures according to conventional processes, e.g., by formation ofdiastereoisomeric salts, by treatment with an optically active acid orbase. Examples of appropriate acids are tartaric, diacetyltartaric,dibenzoyltartaric, ditoluoyltartaric, and camphorsulfonic acid and thenseparation of the mixture of diastereomers by crystallization followedby liberation of the optically active bases from these salts. Adifferent process for separation of optical isomers involves the use ofa chiral chromatography column optimally chosen to maximize theseparation of the enantiomers. Still another method involves synthesisof covalent diastereomers by reacting compounds of the invention with anoptically pure acid in an activated form or an optically pureisocyanate. The synthesized diastereomers can be separated byconventional means such as chromatography, distillation, crystallizationor sublimation, and then hydrolyzed to deliver the enantiomerically purecompound.

Optically active compounds of the invention can be obtained by usingactive starting materials. These isomers may be in the form of a freeacid, a free base, an ester or a salt.

The compounds of this invention can exist in radiolabeled form, i.e.,said compounds may contain one or more atoms containing an atomic massor mass number different from the atomic mass or mass number: ordinarilyfound in nature. Radioisotopes of hydrogen, carbon, phosphorous,fluorine and chlorine include ³H, ¹⁴O ³²P, ³⁵S, ¹⁸F and ³⁶Cl,respectively. Compounds of this invention which contain thoseradioisotopes and/or other radioisotopes of other atoms are within thescope of this invention. Tritiated, i.e., ³H, and carbon-14, i.e., ¹⁴C,radioisotopes are particularly preferred for their ease of preparationand detectability.

Radiolabeled compounds of this invention can generally be prepared bymethods well known to those skilled in the art. Conveniently, suchradiolabeled compounds can be prepared by carrying out the proceduresdisclosed herein except substituting a readily available radiolabeledreagent for a non-radiolabelled reagent.

In a second embodiment, the present invention relates to apharmaceutical composition comprising at least one compound of formula(1) as described above, a salt thereof with a pharmaceuticallyacceptable organic or inorganic acid or base, or an enantiomer thereof,or a N-oxide thereof, or a quaternary ammonium salt thereof, and atleast one pharmaceutically acceptable excipient.

Preferably, the pharmaceutical composition of the present invention isprepared in suitable dosage forms.

Examples of suitable dosage forms are tablets, capsules, coated tablets,granules, solutions and syrups for oral administration; solutions,pomade and ointment for topical administration; medicated patches fortransdermal administration; suppositories for rectal administration andinjectable sterile solutions. Other suitable dosage forms are those withsustained release and those based on liposomes for oral, injectable ortransdermal administration. The pharmaceutical compositions of thisinvention may also be administered by nasal aerosol or inhalation ordelivered by implantation (e.g., surgically), such as with animplantable or indwelling device like a stent.

Other suitable dosage forms are those with sustained release and thosebased on liposomes for oral, injectable or transdermal administration.

The dosage forms of the pharmaceutical composition of the presentinvention can be prepared by techniques that are familiar to apharmaceutical chemist, and comprise mixing, granulation, compression,dissolution, sterilization and the like.

Typically, the amount of compound of formula (1) or of thepharmaceutically acceptable quaternary ammonium salt, N-oxide and saltthereof in the pharmaceutical composition of the present invention willbe between 0.01 mg to 1,500 mg, preferably between 0.1 mg and 500 mg andmore preferably between 1 mg and 200 mg.

Typically, the amount of compound of formula (1) in the pharmaceuticalcomposition of the present invention will be such to ensure a level ofadministration from 0.001 to 20 mg/kg/day. Preferably, the level ofadministration is from 0.01 to 7.5 mg/kg/day, more preferably from 0.1to 5 mg/kg/day, and most preferably from 0.5 to 2.5 mg/kg/day.

As the skilled artisan will appreciate, lower or higher doses than thoserecited above may be required. Specific dosage and treatment regimensfor any particular patient will depend upon a variety of factors,including the activity of the specific compound employed, the age, bodyweight, general health status, sex, diet, time of administration, rateof excretion, drug combination, the severity and course of the disease,and the patient's disposition to the disease and the judgment of thetreating physician.

The pharmaceutical compositions of this invention may be administeredorally, parenterally, by inhalation spray, topically, rectally, nasally,buccally, vaginally or via an implanted reservoir. The term parenteralas used herein includes subcutaneous, intracutaneous, intravenous,intramuscular, intra-articular, intrasynovial, intrasternal,intrathecal, intralesional and intracranial injection or infusiontechniques.

As mentioned above, depending on the nature of the substituents, thecompound of formula (1) may form addition salts with a pharmaceuticallyacceptable organic or inorganic acid or base.

Typical examples of suitable physiologically acceptable inorganic acidsare hydrochloric acid, hydrobromic acid, sulfuric acid, phosphoric acidand nitric acid.

Typical examples of suitable physiologically acceptable organic acidsare acetic acid, ascorbic acid, benzoic acid, citric acid, fumaric acid,lactic acid, maleic acid, methanesulfonic acid, oxalic acid,para-toluenesulfonic acid, benzenesulfonic acid, succinic acid, tannicacid and tartaric acid.

Typical examples of suitable physiologically acceptable inorganic basesare hydroxides, carbonates and hydrogen carbonates of ammonium, calcium,magnesium, sodium and potassium, for instance ammonium hydroxide,calcium hydroxide, magnesium carbonate, sodium hydrogen carbonate andpotassium hydrogen carbonate.

Typical examples of suitable physiologically acceptable organic basesare: arginine, betaine, caffeine, choline, N,N-dibenzylethylenediamine,diethylamine, 2-diethylaminoethanol, 2-dimethylaminoethanol,ethanolamine, ethylenediamine, N-ethylmorpholine, N-ethylpiperidine,N-methylglucamine, glucamine, glucosamine, histidine,N-(2-hydroxyethyl)-piperidine, N-(2-hydroxyethyl)pyrrolidine,isopropylamine, lysine, methylglucamine, morpholine, piperazine,piperidine, theobromine, triethylamine, trimethylamine, tripropylamineand tromethamine.

As described herein, the pharmaceutical composition of the presentinvention comprises a compound of the invention together with apharmaceutically acceptable excipient, which, as used herein, includesany and all solvents, diluents, or other vehicle, dispersion orsuspension aids, surface active agents, isotonic agents, thickening oremulsifying agents, preservatives, solid binders, lubricants and thelike, as suited to the particular dosage form desired.

Some examples of materials which can serve as pharmaceuticallyacceptable excipient include, but are not limited to, sugars such aslactose, glucose and sucrose; starches such as corn starch and potatostarch; cellulose and its derivatives such as sodium carboxymethylcellulose, ethyl cellulose and cellulose acetate; powdered tragacanth;malt; gelatin; talc; excipients such as cocoa butter and suppositorywaxes; oils such as peanut oil, cottonseed oil; safflower oil; sesameoil; olive oil; corn oil and soybean oil; glycols; such a propyleneglycol; esters such as ethyl oleate and ethyl laurate; agar; bufferingagents such as magnesium hydroxide and aluminum hydroxide; alginic acid;pyrogen-free water; isotonic saline; Ringer's solution; ethyl alcohol,and phosphate buffer solutions, other non-toxic compatible lubricantssuch as sodium lauryl sulfate and magnesium stearate, coloring agents,releasing agents, coating agents, sweetening, flavoring and perfumingagents, preservatives and antioxidants.

The terms “pharmaceutically acceptable” and “physiologically acceptable”are intended to define, without any particular limitation, any materialsuitable for preparing a pharmaceutical composition to be administeredto a living being.

In a third embodiment, the present invention relates to the compounds offormula (1) for use in medicine.

In a fourth embodiment, the present invention relates to the compoundsof formula (1) for use in the treatment of bacterial infections.

In a fifth embodiment, the present invention relates to a method fortreating a bacterial infection, comprising the administration of acompound of formula (1) to a patient in need thereof.

Preferably, said bacterial infection is a skin infection, a mucosalinfection, a gynaecological infection, a respiratory tract infection(RTI), a CNS infection, a gastro-intestinal infection, a bone infection,a cardiovascular infection, a sexually transmitted infection, or aurinary tract infection.

More in particular, said bacterial infection is a acute exacerbation ofchronic bronchitis (ACEB), an acute otitis media, an acute sinusitis, aninfection caused by drug resistant bacteria, a catheter-related sepsis,a chancroid, a chlamydia, a community-acquired pneumonia (CAP), acomplicated skin and skin structure infection, an uncomplicated skin andskin structure infection, an endocarditis, a febrile neutropenia, agonococcal cervicitis, a gonococcal urethritis, a hospital-acquiredpneumonia (HAP), a osteomyelitis, a sepsis, a syphilis, aventilator-associated pneumonia, an intraabdominal infections, agonorrhoeae, a meningitis, a tetanus, or a tuberculosis.

Even more, said bacterial infection can be an atherosclerosis orcardiovascular disease related to infection by Helicobacter pylori orChlamydia pneumonia; a blood and tissue infections, includingendocarditis and osteomyelitis, caused by S. aureus, S. haemolyticus, E.faecalis, E. faecium, E. durans, including strains resistant to knownantibacterials such as, but not limited to, beta-lactams, vancomycin,aminoglycosides, quinolones, chloramphenicol, tetracyclines andmacrolides; bronchitis; catheter-related sepsis; chancroid; chlamydia;community-acquired pneumonia; disseminated Mycobacterium avium complex(MAC) disease related to infection by Mycobacterium avium, orMycobacterium intracellulare; endocarditis; febrile neutropenia; gasgangrene related to infection by Clostridium perfringens or Bacteroidesspp; gastroenteritis infection; glomerulonephritis related to infectionby Streptococcus pyogenes, Groups C and G streptococci, Corynebacteriumdiphtheriae, or Actinobacillus haemolyticum; gonococcal cervicitis;gonococcal urethritis; gynaecological infection; hospital-acquiredpneumonia (HAP); infection caused by drug resistant bacteria; infectionscaused by Mycobacterium tuberculosis, M. leprae, M. paratuberculosis, M.kansasii, or M. chelonei; intestinal protozoa related to infection byCryptosporidium spp; Lyme disease related to infection by Borreliaburgdorferi; conjunctivitis, keratitis, and dacrocystitis related toinfection by Chlamydia trachomatis, Neisseria gonorrhoeae, S. aureus, S.pneumoniae, S. pyogenes, H. injluenzae, or Listeria spp.; mastoiditisrelated to infection by Streptococcus pneumoniae, Haemophilusinjluenzae, Moraxella catarrhalis Staphylococcus aureus, Enterococcusfaecalis, E. faecium, E. casseliflavus, S. epidermidis, S. haemolyticus,or Peptostreptococcus spp; odontogenic infection related to infection byviridans streptococci; osteomyelitis; otitis media; persistent coughrelated to infection by Bordetella pertussis; pharyngitis; puerperalfever related to infection by Staphylococcus aureus, coagulase-negativestaphylococci Streptococcus pyogenes, Streptococcus agalactiae,Streptococcal groups C—F (minute colony streptococci), viridansstreptococci Corynebacterium minutissimum, Clostridium spp., orBartonella henselae; respiratory tract infections related to infectionby Mycoplasma pneumoniae, Legionella pneumophila, Streptococcuspneumoniae, Haemophilus injluenzae, or Chlamydia pneumoniae; rheumaticfever; sepsis; sexually transmitted diseases related to infection byChlamydia trachomatis, Haemophilus ducreyi, Treponema pallidum,Ureaplasma urealyticum, or Neiseria gonorrhoeae; sinusitis; syphilis;systemic febrile syndromes related to infection by Borrelia recurrentis;tonsillitis; toxin diseases related to infection by S. aureus (foodpoisoning and toxic shock syndrome), or Groups A, B, and C streptococci;ulcers related to infection by Helicobacter pylori; uncomplicated acuteurinary tract infections related to infection by Staphylococcus aureuscoagulase-negative staphylococcal species, or Enterococcus spp;uncomplicated skin and soft tissue infections and abscesses; urethritisand cervicitis; urinary tract infection; central nervous systeminfections; device related infections caused by staphylococci;muscoleskeletal infection caused by staphylococci; Shiga toxin-producingE. coli; Haemophilus influenzae (invasive disease); legionellosis;psittacosis/ornithosis clamydia psittaci; salmonellosis caused bysalmonella spp; shigellosis by shigella spp; streptococcal toxic shocksyndrome; staphylococcal toxic shock syndrome; and typhoid fever causedby Salmonella typhi.

The bacterial infection can be an infection caused by Acinetobacter spp,Bacteroides spp, Burkholderia spp, Campylobacter spp, Chlamydia spp,Chlamydophila spp, Clostridium spp, Enterobacter spp, Enterococcus spp,Escherichia spp, Gardnerella spp, Haemophilus spp, Helicobacter spp,Klebsiella spp, Legionella spp, Moraxella spp, Morganella spp,Mycoplasma spp, Neisseria spp, Peptostreptococcus spp, Proteus spp,Pseudomonas spp, Salmonella spp, Serratia spp, Staphylococcus spp,Streptoccocus spp, Stenotrophomonas spp, Ureaplasma spp, aerobes,obligate anaerobes, facultative anaerobes, gram-positive bacteria,gram-negative bacteria, gram-variable bacteria, and atypical respiratorypathogens.

More in particular, the bacterial infection can be an infection causedby Acinetobacter baumanii, Acinetobacter haemolyticus, Acinetobacterjunii, Acinetobacter johnsonii, Acinetobacter lwoffi, Bacteroidesbivius, Bacteroides fragilis, Burkholderia cepacia, Campylobacterjejuni, Chlamydia pneumoniae, Chlamydia urealyticus, Chlamydophilapneumoniae, Clostridium difficile, Enterobacter aerogenes, Enterobactercloacae, Enterococcus faecalis, Enterococcus faecium, Escherichia coli,Gardnerella vaginalis, Haemophilus parainfluenzae, Haemophilusinfluenzae, Helicobacter pylori, Klebsiella pneumoniae, Legionellapneumophila, Methicillin-resistant Staphylococcus aureus,Methicillin-susceptible Staphylococcus aureus, Moraxella catarrhalis,Morganella morganii, Mycoplasma pneumoniae, Neisseria gonorrhoeae,Penicillin-resistant Streptococcus pneumoniae, Penicillin-susceptibleStreptococcus pneumoniae, Peptostreptococcus magnus, Peptostreptococcusmicros, Peptostreptococcus anaerobius, Peptostreptococcusasaccharolyticus, Peptostreptococcus prevotii, Peptostreptococcustetradius, Peptostreptococcus vaginalis, Proteus mirabilis, Pseudomonasaeruginosa, Quinolone-Resistant Staphylococcus aureus,Quinolone-Resistant Staphylococcus epidermis, Salmonella typhi,Salmonella paratyphi, Salmonella enteritidis, Salmonella typhimurium,Serratia marcescens, Staphylococcus aureus, Staphylococcus epidermidis,Staphylococcus saprophyticus, Streptococcus agalactiae, Streptococcuspneumoniae, Streptococcus pyogenes, Stenotrophomonas maltophilia,Ureaplasma urealyticum, Vancomycin-Resistant Enterococcus faecium,Vancomycin-Resistant Enterococcus faecalis, Vancomycin-ResistantStaphylococcus aureus, and Vancomycin-Resistant Staphylococcusepidermis.

Examples of compounds according to the present invention are provided inthe following Table 1.

TABLE 1 No. A L₁ Y L₂ R B 157

σ bond

σ bond

160

σ bond

—NH—CH₂—

164

σ bond

σ bond

165

σ bond

σ bond

180

σ bond

—CH₂—NH—CH₂— σ bond

181

σ bond

—CH₂—NH—CH₂— σ bond

182

σ bond

—CH₂—NH—CH₂— σ bond

183

σ bond

—CH₂—NH—CH₂— σ bond

193

σ bond

—NH—CH₂— σ bond

194

σ bond

—CH₂—CH₂— σ bond

197

σ bond

—CH₂—CH₂— σ bond

200

σ bond

—CH₂—CH₂— σ bond

201

σ bond

—NH—CH₂— σ bond

202

σ bond

—CH₂—CH₂— σ bond

204

σ bond

—NH—CH₂— σ bond

205

σ bond

—NH—CH₂— σ bond

206

σ bond

—NH—CH₂— σ bond

207

σ bond

—NH—CH₂— σ bond

208

σ bond

—NH—CH₂— σ bond

209

σ bond

—NH—CH₂— σ bond

210

σ bond

—NH—CH₂— σ bond

211

σ bond

—NH—CH₂— σ bond

212

σ bond

—NH—CH₂— σ bond

213

σ bond

—NH—CH₂— σ bond

214

σ bond

—NH—CH₂— σ bond

215

σ bond

—NH—CH₂— σ bond

216

σ bond

—NH—CH₂— σ bond

217

σ bond

—NH—CH₂— σ bond

219

σ bond

—NH—CH₂— σ bond

220

σ bond

—NH—CH₂— σ bond

221

σ bond

σ bond

301

σ bond

—NH—CH₂— σ bond

302

σ bond

—NH—CH₂— σ bond

303

σ bond

σ bond

304

σ bond

σ bond

305

σ bond

—NH—CH₂— σ bond

306

σ bond

—NH—CH₂— σ bond

307

σ bond

σ bond

308

σ bond

σ bond

309

σ bond

—NH—CH₂— σ bond

310

σ bond

—NH—CH₂— σ bond

311

σ bond

—CH₂—NH—CH₂— σ bond

312

σ bond

σ bond

313

σ bond

—CH₂—NH—CH₂— σ bond

314

σ bond

—NH—CH₂— σ bond

315

σ bond

—NH—CH₂— σ bond

316

σ bond

—NH—CH₂—

317

—CH₂—

—NH—CH₂— σ bond

318

—CH₂— σ bond

319

σ bond

—NH—CH₂— σ bond

320

σ bond

—NH—CH₂— σ bond

321

—CH₂—

—CH₂— σ bond

322

σ bond

—NH—(CH₂)₂—NH— σ bond

323

σ bond

σ bond

324

σ bond

—NH—CH₂— σ bond

325

σ bond

—NH—CH₂— σ bond

326

σ bond

—NH—CH₂—

327

—CH₂— σ bond

328

σ bond σ bond

329

σ bond σ bond

330

σ bond

—NH—CH₂— σ bond

331

σ bond

—NH—CH₂— σ bond

332

σ bond

—NH—CH₂— σ bond

333

σ bond

—CH₂—CH₂— σ bond

334

σ bond

—CH₂—CH₂— σ bond

335

σ bond

—CH₂—CH₂— σ bond

336

σ bond

—CH₂—CH₂— σ bond

337

σ bond

—CH₂—CH₂—

338

σ bond

—CH₂—CH₂—

339

σ bond

—NH—CH₂— σ bond

340

—CH₂—

—CH₂— σ bond

341

—CH₂—

—CH₂— σ bond

342

—CH₂—

—NH— σ bond

343

—CH₂—

σ bond

344

—CH₂—

σ bond

345

—CH₂—

σ bond

347

—CH₂—

σ bond

348

σ bond

σ bond

349

σ bond

σ bond

350

σ bond

σ bond

351

σ bond

σ bond

352

σ bond

σ bond

354

σ bond

σ bond

The above compounds can be prepared as explained in the syntheticexamples below.

The man skilled in the art has a well-established literature ofheterocyclic and other relevant chemical transformations, recovery andpurification technologies to draw upon, in combination with theinformation contained in the examples which follow, for guidance onsynthetic strategies, protecting groups, and other materials and methodsuseful for the synthesis, recovery and characterization of the compoundsof this invention, including compounds containing the various choicesfor A, L₁, Y, L₂, R and B.

Various synthetic approaches may be used to produce the compoundsdescribed herein, including those approaches depicted schematicallybelow. The man skilled in the art will appreciate that protecting groupsmay be used in these approaches. “Protecting groups”, are moieties thatare used to temporarily block chemical reaction at a potentiallyreactive site (e.g., an amine, hydroxy, thiol, aldehyde, etc.) so that areaction can be carried out selectively at another site in amultifunctional compound. In preferred embodiments, a protecting groupreacts selectively in good yield to give a protected substrate that issuitable for the planned reactions; the protecting group should beselectively removable in good yield by readily available, preferablynontoxic reagents that do not unduly attack the other functional groupspresent; the protecting group preferably forms an readily separablederivative (more preferably without the generation of new stereogeniccenters); and the protecting group preferably has a minimum ofadditional functionality to avoid the complication of further sites ofreaction. A wide variety of protecting groups and strategies, reagentsand conditions for deploying and removing them are known in the art.

Also, one may chose reagents enriched for a desired isotope, e.g.tritium in place of hydrogen, to create compounds of this inventioncontaining such isotope(s). Compounds containing tritium in place ofhydrogen in one or more locations, or containing various isotopes of C,N, P and O, are encompassed by this invention and may be used, forinstance, for studying metabolism and/or tissue distribution of thecompounds or to alter the rate or path of metabolism or other aspects ofbiological functioning.

The compounds of the this invention can be synthesized using the methodsdescribed below, together with synthetic methods known in the art ofsynthetic organic chemistry, or by a variation thereon as appreciated bythose skilled in the art. Preferred methods include, but are not limitedto those described below. The reactions are performed in a solventappropriate to the reagents and materials employed and suitable for thetransformation being effected. It will be understood by those skilled inthe art of organic synthesis that the functionality present on themolecule should be consistent the transformations proposed. This willsometimes required some judgment to modify the order of the syntheticsteps or to select one particular process scheme over another in orderto obtain a desired compound of the invention.

A compound of the present invention could be prepared as outlined in thesynthetic pathways described hereinafter and via standard methods knownto those skilled in the art.

EXAMPLES

List of the abbreviations used in the synthetic pathways describedhereinafter:

Boc: tert-butyl carbamate

cHex cyclohexane

CV column volume

DBU: 1,5-diazabiciclo[5.4.0]undec-5-ene

DCM: dichloromethane

DIPEA N,N-diisopropylethylamine

DME: 1,2-dimethoxyethane

DMF: N,N-dimethylformamide

Et₂O: diethyl ether

EtOAc: ethylacetate

MS: mass spectroscopy

TEA: triethylamine

TFA: trifluoroacetic acid

THF: tetrahydrofuran

Pd/C: palladium on activated charcoal

Pd(OH)₂/C: palladium hydroxide on activated charcoal

r.t.: room temperature

UPLC: Ultra High Performance Liquid Chromatography

Preparation of Compound 157

Compound 157 was prepared as described herein below.

Step 1—Synthesis of methyl 5-fluoro-2-hydroxybenzoate

To a stirred solution of 5-fluoro salicylic acid (25 g, 160 mmol) inMeOH (250 mL), conc. sulfuric acid (20 mL) was added slowly at 0° C.

The resulting reaction mixture was refluxed for 48 h then wasconcentrated under reduced pressure and the resulting crude was basifiedto pH 8.0 with sat. NaHCO₃. The mixture was neutralized by 1.5 N HClsolution and extracted with EtOAc. The organic layer was dried overanhydrous Na₂SO₄ and concentrated to afford methyl5-fluoro-2-hydroxybenzoate as a light brown liquid (22.8 g, 83% yield).GC-MS (AcqMethod HP-1MS.M): 170.1 (M). ¹H NMR (400 MHz, DMSO-d6): δ ppm10.29 (s, 1H), 7.51-7.49 (m, 1H), 7.42-7.41 (m, 1H), 7.03-7.01 (m, 1H),3.89 (s, 3H).

Step 2—Synthesis of 5-fluoro-2-hydroxybenzamide

A mixture of methyl 5-fluoro-2-hydroxybenzoate (22 g, 129 mmol) andmethanolic ammonia (250 mL) was heated at 50° C. in an autoclave for 10h. The reaction mixture was concentrated under reduced pressure, theresulting crude was codistilled with toluene and dried to give5-fluoro-2-hydroxybenzamide as a brown solid (18.5 g, 92% yield). LC-MSm/z: 154.0 (M−H+). ¹H NMR (400 MHz, DMSO-d6): δ ppm 12.74 (s, 1H), 8.40(s, 1H), 8.03 (s, 1H), 7.73-7.71 (m, 1H), 7.31-7.29 (m, 1H), 6.91-6.90(m, 1H).

Step 3—Synthesis of 6-fluoro-2H-1,3-benzoxazine-2,4(3H)-dione

To a stirred solution of 5-fluoro-2-hydroxybenzamide (8.0 g, 51.6 mmol)in dry THF (80 mL), 1,1′-carbonyldiimidazole (10.9 g, 67.09 mmol) wasadded at 0° C. The mixture was stirred at room temperature for 14 h thenwas concentrated under reduced pressure. The resulting crude was treatedwith MeOH and washed with diethyl ether. The resulting white solid wasdried and used in the next step without further purification (5.1 g, 55%yield, white solid). LC-MS m/z: 180.0 (M−H⁺). ¹H NMR (400 MHz, DMSO-d6):δ ppm 12.19 (s, 1H), 7.68-7.67 (m, 2H), 7.50-7.48 (m, 1H).

Step 4—Synthesis of 4-chloro-6-fluoro-2H-1,3-benzoxazin-2-one

To a stirred solution of 6-fluoro-2H-1,3-benzoxazine-2,4(3H)-dione (0.5g, 2.76 mmol) in dry 1,2-dichloroethane (2.5 mL), phosphorouspentachloride (0.69 g, 3.31 mmol) was added at 0° C. The resultingmixture was refluxed for 6 h then was concentrated under reducedpressure. DCM (15 mL) was added to the resulting crude, washed withwater (2 mL) and dried over anhydrous Na₂SO₄. The solvent wasconcentrated under reduced pressure to afford4-chloro-6-fluoro-2H-1,3-benzoxazin-2-one as an off-white solid (0.46 g,84% yield). ¹H NMR (400 MHz, DMSO-d6): δ ppm 7.69-7.67 (m, 2H),7.50-7.49 (m, 1H).

Step 5—Synthesis of tert-butyl4-(3-chloro-2-hydroxypropyl)piperazine-1-carboxylate

To a solution of tert-butyl piperazine-1-carboxylate (6.0 g, 32.2 mmol)in ethanol (100 mL), epichlorohydrine (14.8 g, 161.0 mmol) was added andthe solution was stirred for 15 h at room temperature. After completionof the reaction, the mixture was concentrated under reduced pressure andthe resulting crude product (colorless thick liquid, 8.9 g, 99% yield)was used as such in the following step. LC-MS m/z: 279.2 (M+1). ¹HNMR(400 MHz, DMSO-d6): δ 1.40 (s, 9H), 2.27-2.42 (m, 6H), 3.21-3.32 (m,4H), 3.52-3.56 (m, 1H), 3.62-3.67 (m, 1H), 3.79-3.89 (m, 1H), 5.04 (d,J=4.9 Hz, 1H).

Step 6—Synthesis of tert-butyl 4-(3-azido-2-hydroxypropyl)piperazine-1-carboxylate

To a solution of tert-butyl4-(3-chloro-2-hydroxypropyl)piperazine-1-carboxylate (8.9 g, 32 mmol) inDMF (80 mL) sodium azide (3.12 g, 48 mmol) was added. The mixture wasstirred at 85° C. for 5 h then was diluted with ethyl acetate (300 mL)and washed with water. The organic layer was dried over sodium sulfateand concentrated under reduced pressure. The resulting crude waspurified on silica gel (60-120 mesh) column chromatography (0-10%methanol in dichloromethane) to obtain tert-butyl4-(3-azido-2-hydroxypropyl)piperazine-1-carboxylate (8.2 g, 89% yield)as a pale yellow thick liquid. LC-MS: 286.2 (M+1). ¹HNMR (400 MHz,DMSO-d6): δ 1.40 (s, 9H), 2.29-2.42 (m, 6H), 3.15-3.20 (m, 1H),3.24-3.32 (m, 5H), 3.79-3.82 (m, 1H), 5.04 (d, J=4.9 Hz, 1H).

Step 7—Synthesis of tert-butyl 4-(3-amino-2-hydroxypropyl)piperazine-1-carboxylate

To a solution of tert-butyl4-(3-azido-2-hydroxypropyl)piperazine-1-carboxylate (1.5 g, 5.2 mmol) inethanol (40 mL), 10% palladium on carbon (0.15 g) was added and themixture was stirred under hydrogen atmosphere at room temperature for 3h. The reaction mixture was filtered through a celite bed and thefiltrate was concentrated under reduced pressure to obtain tert-butyl4-(3-amino-2-hydroxypropyl) piperazine-1-carboxylate (1.25 g, 85% yield)as a pale yellow color solid. LC-MS m/z: 260.6 (M+1). ¹HNMR (300 MHz,DMSO-d6): δ 1.39 (s, 9H), 2.22-2.43 (m, 8H), 2.58-2.66 (m, 2H),3.48-3.52 (m, 4H), 3.88-3.90 (m, 1H).

Step 8—Synthesis of tert-butyl4-(3-((6-fluoro-2-oxo-2H-benzo[e][1,3]oxazin-4-yl)amino)-2-hydroxypropyl)piperazine-1-carboxylate

To a stirred solution of 4-chloro-6-fluoro-2H-benzo[e][1,3]oxazin-2-one(0.89 g, 4.5 mmol) and DIPEA (2.9 g, 22.5 mmol) in acetonitrile (50 mL),tert-butyl 4-(3-amino-2-hydroxypropyl)piperazine-1-carboxylate (1.39 g,5.4 mmol) was added at room temperature. The mixture was stirred at thesame temperature overnight. The solid was filtered, washed with waterand dried. The crude product was purified on silica gel columnchromatography (0-20% methanol in dichloromethane) to obtain the titlecompound (0.4 g, 21% yield) as an off-white solid. LC-MS m/z: 423.2(M+1). ¹HNMR (300 MHz, DMSO-d6): δ 1.38 (s, 9H), 2.27-2.50 (m, 6H),3.28-3.39 (m, 4H), 3.66-3.71 (m, 1H), 3.98 (br. S, 1H), 4.97 (br s, 1H),7.35-7.40 (m, 1H), 7.59-7.65 (m, 1H) 8.07-8.10 (d, J=6.3 Hz, 1H),9.10-9.11 (m, 1H).

Step 9—Synthesis of6-fluoro-4-((2-hydroxy-3-(piperazin-1-yl)propyl)amino)-2Hbenzo[e][1,3]oxazin-2-onehydrochloride

A solution of tert-butyl4-(3-((6-fluoro-2-oxo-2H-benzo[e][1,3]oxazin-4yl)amino-2-hydroxypropyl)piperazine-1-carboxylate (100 mg, 0.023 mmol) inHCl.dioxane (4.5 N solution, 5 mL) was stirred for 4 h at roomtemperature. The reaction mixture was concentrated under reducedpressure and the resulting product (off-white solid, 100 mg, quant.yield) was used as such in the next step.

Step 10—Synthesis of6-fluoro-4-({2-hydroxy-3-[4-(2,7-naphthyridin-1-yl)piperazin-1-yl]propyl}amino)-2H-1,3-benzoxazin-2-one(compound 157)

To a solution of6-fluoro-4-((2-hydroxy-3-(piperazin-1-yl)propyl)amino)-2Hbenzo[e][1,3]oxazin-2-onehydrochloride (200 mg, 0.56 mmol) and TEA (0.18 mL, 0.85 mmol) in DMF (2mL), 1-chloro-2,7-naphthyridine (60 mg, 0.37 mmol) was added. Thereaction mixture was stirred for 8 h at 80° C. then was cooled andconcentrated under reduced pressure. The resulting crude was purified byflash column chromatography (5 to 15% MeOH in DCM) to give the titlecompound as a light brown solid (35 mg, 14% yield). LC-MS m/z: 451.2(M+1). ¹HNMR (DMSO d6): 2.49-2.78 (m, 4H), 3.07-3.31 (m, 6H), 3.02-3.04(m, 2H), 4.37-4.38 (m, 1H), 6.08 (s, 1H) 7.32-7.49 (m, 2H), 7.48-7.49(m, 1H), 7.65-7.71 (m, 1H), 8.12-8.18 (m, 1H), 8.31-8.32 (m, 1H),8.64-8.66 (m, 1H), 9.32-9.43 (m, 1H), 9.93-9.94 (m, 1H).

Preparation of Compound 160

Compound 160 was prepared as described herein below.

Step 1—Synthesis of tert-butyl[1-(7-fluoroisoquinolin-1-yl)piperidin-4-yl]carbamate

Potassium carbonate (2.9 g, 21 mmol) was added to a stirred solution of1-chloro-7-fluoroisoquinoline (2.6, 14 mmol) and tert-butylpiperidin-4-ylcarbamate (5.2 g, 28 mmol) in DMSO (20 mL) at roomtemperature. The resulting mixture was stirred at 120° C. overnight thenwas allowed to cool to room temperature and partitioned between EtOAc(300 mL) and water (300 mL). The organic layer was separated, washedwith 1M citric acid solution (100 mL) and brine (70 mL) and dried oversodium sulfate. The solvents were evaporated under reduced pressure andthe resulting residue was purified by flash chromatography on silica gel(SNAP 100, from Cy to Cy/Ethyl acetate 8:2) to obtain tert-butyl[1-(7-fluoroisoquinolin-1-yl)piperidin-4-yl]carbamate (4.3 g, 12.4 mmol,88% yield). LC-MS (M−H⁺)=346.5

Step 2—Synthesis of 1-(7-fluoroisoquinolin-1-yl)piperidin-4-amine

TFA (10 mL) was added to a solution of tert-butyl4-(7-fluoroisoquinolin-1-yl)piperazine-1-carboxylate (4.3 g, 12.4 mmol)in dichloromethane (30 mL) and the resulting mixture was stirredovernight at room temperature. The volatiles were evaporated underreduced pressure, the residue was dissolved in dichloromethane (20 mL)and evaporated under reduced pressure. The resulting residue wasdissolved in MeOH and loaded onto a preconditioned SCX cartridge (50 g).The SCX was eluted with MeOH and a 2M solution of ammonia in methanol.The basic fraction was evaporated under reduced pressure to give 3.1 g(Y=quant.) of 1-(7-fluoroisoquinolin-1-yl)piperidin-4-amine. LC-MS(M−H⁺)=246.3

Step 3—Synthesis of 2-(pyridin-2-yl)-1H-imidazole-4-carbaldehyde

To a suspension of [2-(pyridin-2-yl)-1H-imidazol-4-yl]methanol (1 g, 5.7mmol) in dry DCM (30 mL) Dess-Martin Periodinane (3.1 g, 1.3 eq) wasadded portionwise. The cloudy mixture was stirred 1.5 h at roomtemperature then was treated with sat. NaHCO₃ and 10% Na₂S₂O₃ solution.After stirring for 30 min. the organic phase was separated, dried overNa₂SO₄, filtered and evaporated to recover 730 mg of crude2-(pyridin-2-yl)-1H-imidazole-4-carbaldehyde, that was progressedwithout further purification and characterization.

Step 4—Synthesis of1-(7-fluoroisoquinolin-1-yl)-N-{[2-(pyridin-2-yl)-1H-imidazol-4-yl]methyl}piperidin-4-amine(formate salt, compound 160)

2-(pyridin-2-yl)-1H-imidazole-4-carbaldehyde (100 mg, 0.57 mmol) wasdissolved in dry DCM (20 mL) and treated with1-(7-fluoroisoquinolin-1-yl)piperidin-4-amine (140 mg, 0.57 mmol) and 2drops of acetic acid. After stirring for 30 min. NaBH(OAc)₃ (180 mg,0.86 mmol) was added in one portion and the reaction mixture was stirredovernight. The reaction was partitioned between DCM and sat. NaHCO₃. Theorganic phase was evaporated and purified by reversed phasechromatography (H₂O/MeCN+0.1% HCOOH from 100/0 to 85/15) to afford1-(7-fluoroisoquinolin-1-yl)-N-{[2-(pyridin-2-yl)-1H-imidazol-4-yl]methyl}piperidin-4-amineas formate salt (56 mg, 0.125 mmol, 21.9% yield). LC-MS (M−H⁺)=403.4. ¹HNMR (500 MHz, METHANOL-d₄) ppm 1.95-2.08 (m, 2H), 2.35 (dd, J=11.74,1.47 Hz, 2H), 3.06 (t, J=12.00 Hz, 2H), 3.40-3.50 (m, 1H), 3.86 (d,J=12.00 Hz, 2H), 4.34 (s, 2H), 7.37-7.41 (m, 1H), 7.41-7.47 (m, 2H),7.55 (td, J=8.80, 2.45 Hz, 1H), 7.75 (dd, J=10.27, 2.45 Hz, 1H),7.86-7.99 (m, 2H), 8.06-8.11 (m, 2H), 8.44 (s, 1H), 8.63 (d, J=4.40 Hz,1H).

Preparation of Compound 164

Compound 164 was prepared as described herein below.

Step 1—Synthesis of 8-chloro-2,7-naphthyridine 2-oxide

To a solution of 1-chloro-2,7-naphthyridine (0.15 g, 0.9 mmol) in DCM,m-CPBA (55%, 472 mg, 1.4 mmol) was added. The mixture was stirred 3 h atroom temperature then the organic layer was washed with saturatedpotassium carbonate solution, water, brine solution and dried oversodium sulfate. The solvent was removed under reduced pressure to give8-chloro-2,7-naphthyridine 2-oxide (120 mg, 73%) as a pale yellow solid.LC-MS m/z: 181.0 (M+1). ¹HNMR (400 MHz, DMSO d6): δ 7.94 (d, J=5.6 Hz,1H), 8.11 (d, J=7.12 Hz, 1H), 8.43-8.39 (m, 1H), 8.49-8.46 (m, 1H), 8.95(s, 1H).

Step 2—Synthesis of8-(4-(3-((6-fluoro-2-oxo-2H-benzo[e][1,3]oxazin-4-yl)amino)-2-hydroxypropyl)piperazin-1-yl)-2,7-naphthyridine2-oxide (compound 164)

The synthesis was performed according to the procedure described forcompound 157 (step 10) using 8-chloro-2,7-naphthyridine 2-oxide (23%yield). LC-MS m/z: 467.0 (M+1). ¹HNMR (400 MHz, DMSO-d6): δ 2.74-2.60(m, 5H), 3.16-3.09 (m, 1H), 3.42-3.21 (m, 5H), 3.72-3.70 (m, 1H),4.06-4.05 (m, 1H), 5.01 (d, J=4.4 Hz, 1H), 7.39-7.36 (m, 2H), 7.65-7.60(m, 1H), 7.91 (d, J=6.8 Hz, 1H), 8.13-8.10 (m, 1H), 8.18 (d, J=5.6 Hz,1H), 8.31 (d, J=7.2 Hz, 1H), 8.64 (s, 1H), 9.14-9.12 (m, 1H).

Preparation of Compound 165

Compound 165 was prepared as described herein below.

Step 1—Synthesis of4-(3-((6-fluoro-2-oxo-2H-benzo[e][1,3]oxazin-4-yl)amino)-2-hydroxypropyl)-1-(2,7-naphthyridin-1-yl)piperazine1-oxide (compound 165)

To a solution of compound 157 (30 mg, 0.06 mmol) in IPA (3 mL),magnesium monoperoxyphthalate hexahydrate (32 mg, 0.06 mmol) was addedat room temperature. The reaction mixture was refluxed for 6 h then wascooled and concentrated under reduced pressure. The resulting crude waspurified by flash column chromatography (8 to 10% MeOH/NH₃ in DCM) togive the title compound (3.5 mg, 11.2% yield). LC-MS m/z: 465.2 (M−1).¹HNMR (CDCl₃+MeOH-d4): δ 1.28 (t, J=6.8 Hz, 3H), 3.35-3.29 (m, 2H),3.71-3.56 (m, 5H), 3.86-3.78 (m, 2H), 4.03-3.98 (m, 2H), 4.61-4.59 (m,1H), 7.22-7.20 (m, 2H), 7.35-7.30 (m, 1H), 7.58-7.56 (m, 1H), 7.70 (d,J=6 Hz, 1H), 8.22 (d, J=5.6 Hz, 1H), 8.53 (d, J=4.8 Hz, 1H), 9.31 (s,1H).

Preparation of Compounds 180 and 181

Compounds 180 and 181 were prepared as described herein below.

Synthesis of4-oxo-1,4-dihydro[1]benzopyrano[4,3-b]pyrrole-2-carbaldehyde

The title intermediate was prepared according to the procedure describedfor the synthesis of8-fluoro-4-oxo-1,4-dihydro[1]benzopyrano[4,3-b]pyrrole-2-carbaldehyde(compound 193, steps 1-4) starting from 4-hydroxy-2H-1-benzopyran-2-one.LC-MS m/z: 212.0 (M−H⁻). ¹HNMR (DMSO d6): δ 12.61 (s, 1H), 9.76 (s, 1H),8.38-8.36 (m, 1H), 7.70 (d, J=2.0 Hz, 1H), 7.61 (d, J=1.6, 1H),7.43-7.41 (m, 2H).

Step 1—Synthesis of tert-butyl 3-hydroxypyrrolidine-1-carboxylate

To a stirred solution of tert-butyl 3-oxopyrrolidine-1-carboxylate (5.2g, 26.9 mmol) in MeOH/THF 1:1 (50 mL), sodium borohydride (2.05 g, 53.9mmol) was added portionwise at 0° C. The reaction mixture was stirredfor 40 min at room temperature then was quenched with ice. The solventwas concentrated under reduced pressure, the resulting residue wasdiluted with EtOAc (100 mL) and washed with water (50 mL) and brine (50mL). The organic layer was dried over sodium sulfate and concentrated toafford the title compound (5.1 g, 98% yield) as a mixture of isomers.¹HNMR (CDCl₃): δ 4.86 (d, J=4.8 Hz, 1H), 4.20 (s, 1H), 2.25-2.23 (m,3H), 3.11-3.01 (m, 1H), 1.83-1.81 (m, 2H), 1.39 (s, 9H).

Step 2—Synthesis of tert-butyl3-[(methanesulfonyl)oxy]pyrrolidine-1-carboxylate

To a stirred solution of tert-butyl 3-hydroxypyrrolidine-1-carboxylate(mixture of isomers, 5.1 g, 27.2 mmol) in DCM (75 mL), TEA (5.9 mL, 42.7mmol) was added at room temperature. The mixture was cooled to 0° C.then methane sulfonyl chloride (2.62 mL, 32.7 mmol) was added dropwise.The resulting solution was stirred at room temperature for 1 h then wasdiluted with DCM (100 mL) and washed with water (50 mL) and brine (50mL). The organic layer was dried over sodium sulfate and concentrated toafford the title compound (7.15 g, 99% yield) as a mixture of isomers,that was progressed without any further purification. ¹HNMR (CDCl₃): δ5.30-5.27 (m, 1H), 3.60-3.58 (m, 4H), 3.05 (s, 3H), 2.19-2.16 (m, 1H),2.13-2.12 (m, 1H), 1.39 (s, 9H).

Step 3—Synthesis of tert-butyl 3-cyanopyrrolidine-1-carboxylate

To a stirred solution of tert-butyl3-[(methanesulfonyl)oxy]pyrrolidine-1-carboxylate (mixture of isomers,6.1 g, 23.0 mmol) in DMF (40 mL) NaCN (3.38 g, 69.0 mmol) was added. Thereaction mixture was stirred at 100° C. for 16 h then was cooled to roomtemperature and diluted with EtOAc (75 mL). The organic layer was washedwith water (2×40 mL), dried over sodium sulfate and concentrated. Theresulting crude was purified by column chromatography (silica gel:230-400, 25% EtOAc in pet. ether) to afford the title compound (3.1 g,15.8 mmol, 69% yield, colorless liquid) as a mixture of isomers. ¹HNMR(CDCl₃): δ 3.69-3.59 (m, 4H), 3.12-3.09 (m, 1H), 2.27-2.25 (m, 2H), 1.48(s, 9H).

Step 4—Synthesis of tert-butyl 3-(aminomethyl)pyrrolidine-1-carboxylate

To a stirred solution of tert-butyl 3-cyanopyrrolidine-1-carboxylate(mixture of isomers, 3.1 g, 15.8 mmol) and TEA (1.3 mL, 9.4 mmol) inEtOH (90 mL) Raney nickel (2.04 g, 23.7 mmol) was added under nitrogen.The reaction mixture was stirred at room temperature for 16 h under 30psi hydrogen pressure then was filtered through celite washing withethanol (50 mL). The solution was concentrated under reduced pressure toafford the title compound (2.6 g, 82% yield, yellow liquid) as a mixtureof isomers. LC-MS (ELSD) m/z: 201.2 (M+H⁺). ¹HNMR (CDCl₃): δ 3.49-3.41(m, 4H), 3.00-2.99 (m, 1H), 2.73 (m, 2H), 2.23-2.22 (m, 1H), 2.00 (m,1H), 1.46 (s, 9H).

Step 5—Synthesis of tert-butyl3-({[(benzyloxy)carbonyl]amino}methyl)pyrrolidine-1-carboxylate

To a stirred solution of tert-butyl3-(aminomethyl)pyrrolidine-1-carboxylate (mixture of isomers, 2.5 g,12.4 mmol) in 1,4 dioxane (50 mL) a saturated sodium carbonate solution(14 mL) was added at room temperature. The reaction mixture was cooledto 0-5° C. then benzyl chloroformate (50% in toluene, 5.5 mL, 16 mmol)was slowly added. After stirring for 5 h at room temperature thereaction mixture was diluted with water (50 mL) and extracted with EtOAc(3×60 mL). The combined organic layers were washed with brine (50 mL),dried over sodium sulfate and evaporated under vacuum. The crude waspurified by column chromatography (Silica gel: 200-400, 12-15% EtOAc inpet. ether) to afford the title compound (2.4 g, 57.5% yield, colorlessliquid) as a mixture of isomers. LC-MS m/z: 235.1 (M+H⁺-Boc). ¹HNMR(CDCl₃): δ 7.36-7.27 (m, 5H), 5.11 (s, 2H), 4.87-4.85 (m, 1H), 3.50-3.05(m, 6H), 3.00-2.97 (m, 1H), 2.35-2.20 (m, 1H), 2.05-1.97 (m, 1H), 1.46(s, 9H).

Step 6—Synthesis of benzyl [(pyrrolidin-3-yl)methyl]carbamatehydrochloride

To a stirred solution of tert-butyl3-({[(benzyloxy)carbonyl]amino}methyl)pyrrolidine-1-carboxylate (mixtureof isomers, 2.4 g, 7.1 mmol) in 1,4-dioxane (30 mL), HCl (4 N solutionin 1,4-dioxane, 30 mL) was added at 0-5° C. The reaction mixture wasstirred for 2 h at room temperature then was concentrated under reducedpressure. The resulting crude was treated with petroleum ether, filteredand dried under reduced pressure to afford the title compound (1.7 g,crude) as a mixture of isomers, that was progressed without any furtherpurification. LC-MS m/z: 235.2 (M−H⁺).

Step 7—Synthesis of benzyl{[1-(7-fluoroisoquinolin-1-yl)pyrrolidin-3-yl]methyl}carbamate

To a stirred solution of benzyl [(pyrrolidin-3-yl)methyl]carbamatehydrochloride (mixture of isomers, 957 mg, 3.5 mmol) in NMP (15 mL) TEA(1.1 g, 11.0 mmol) was added. After stirring for 15 min at roomtemperature, 1-chloro-7-fluoroisoquinoline (400 mg, 2.2 mmol) was added.The reaction mixture was stirred at 140° C. for 24 h then was cooled,concentrated under reduced pressure and diluted with EtOAc (50 mL). Theorganic phase was washed with water (2×25 mL) and brine (20 mL), driedover sodium sulfate and evaporated under vacuum. The resulting crude waspurified by column chromatography (silica gel: 200-400 mesh, 30-40%EtOAc in pet. ether) to afford the title compound (500 mg, 60% yield) asa mixture of isomers. LC-MS m/z: 380.2 (M+H).

Step 8—Synthesis of1-[1-(7-fluoroisoquinolin-1-yl)pyrrolidin-3-yl]methanamine

To a stirred solution of benzyl{[1-(7-fluoroisoquinolin-1-yl)pyrrolidin-3-yl]methyl}carbamate (mixtureof isomers, 500 mg, 1.3 mmol) in methanol (50 mL), 10% Pd/C (100 mg, 50%wet) was added. The reaction mixture was stirred under hydrogenatmosphere for 3 h then was filtered onto a celite bed washing withmethanol (50 mL). The filtrate was concentrated under reduced pressureto afford the title compound (310 mg, 1.3 mmol, 97% yield) as a mixtureof isomers. LC-MS m/z: 246.1 (M+H).

Step 9—Synthesis of the enantiomers of2-[({[1-(7-fluoroisoquinolin-1-yl)pyrrolidin-3-yl]methyl}amino)methyl][1]benzopyrano[4,3-b]pyrrol-4(1H)-one(compounds 180 and 181)

To a stirred solution of1-[1-(7-fluoroisoquinolin-1-yl)pyrrolidin-3-yl]methanamine (mixture ofisomers, 320 mg, 1.3 mmol) and 4-oxo-1, 4-dihydrochromeno [4, 3-b]pyrrole-2-carbaldehyde (330 mg, 1.56 mmol) in DCE (5 mL) and DMF (2 mL),AcOH (0.1 mL) was added at room temperature. After stirring for 20 h at50° C. the mixture was cooled to room temperature and sodium triacetoxyborohydride (553 mg, 2.6 mmol) was added. The reaction mixture wasstirred for 3 h at room temperature then was diluted with water (30 mL)and extracted with DCM (3×30 mL). The combined organic layers werewashed with water (2×20 mL) and brine (20 mL), dried over sodium sulfateand concentrated under reduced pressure. The resulting crude waspurified by flash chromatography (silica gel: 200-400, 3-5% MeOH in DCM)to afford the isomeric mixture of2-[({[1-(7-fluoroisoquinolin-1-yl)pyrrolidin-3-yl]methyl}amino)methyl][1]benzopyrano[4,3-b]pyrrol-4(1H)-one.The mixture was purified by chiral HPLC to afford the 2 enantiomers.

Compound 180 (stereochemistry not assigned): 29.2 mg, 5% yield. LC-MSm/z: 443.1 (M+H). ¹H NMR (400 MHz, DMSO-d6): δ 12.56 (brs, 1H), 8.06 (d,J=7.56 Hz, 1H), 7.93-7.80 (m, 3H), 7.55-7.50 (m, 1H), 7.45-7.36 (m, 2H),7.34-7.32 (m, 1H), 7.05 (d, J=5.6 Hz, 1H), 6.55 (s, 1H), 3.85-3.69 (m,5H), 3.58 (t, J=9.6 Hz, 1H), 2.66-2.50 (m, 2H), 2.43-2.41 (m, 2H),2.09-2.06 (m, 1H), 1.70-1.68 (m, 1H).

Compound 181 (stereochemistry not assigned): 21.4 mg, 3.7% yield. LC-MSm/z: 443.1 (M+H⁺). ¹H NMR (400 MHz, DMSO-d6): δ 12.46 (brs, 1H), 8.05(d, J=7.76 Hz, 1H), 7.93-7.82 (m, 3H), 7.53 (t, J=8.5 Hz, 1H), 7.46-7.33(m, 3H), 7.05 (d, J=5.2 Hz, 1H), 6.54 (s, 1H), 3.82-3.71 (m, 5H), 3.59(t, J=9.08 Hz, 1H), 3.01-3.00 (m, 2H), 2.66-2.61 (m, 1H), 2.43-2.32 (m,1H), 2.07-2.05 (m, 1H), 1.70-1.68 (m, 1H).

Preparation of Compounds 182 and 183

Compounds 182 and 183 were prepared as described herein below.

Step 1—Synthesis of tert-butyl2-{[(methanesulfonyl)oxy]methyl}morpholine-4-carboxylate

The title intermediate was prepared as a mixture of isomers according tothe procedure described for the synthesis of tert-butyl3-[(methanesulfonyl)oxy]pyrrolidine-1-carboxylate (see compounds180-181, step 2) using tert-butyl2-(hydroxymethyl)morpholine-4-carboxylate (92% yield). LC-MS (ELSD) m/z:196.0 (M+H⁺-Boc). ¹HNMR (CDCl₃): δ 4.25 (d, J=4.8 Hz, 2H), 3.93-3.73 (m,3H), 3.69-3.67 (m, 1H), 3.58-3.55 (m, 1H), 3.10 (s, 3H), 2.98-2.85 (m,1H), 2.80-2.75 (m, 1H), 1.43 (s, 9H).

Step 2—Synthesis of tert-butyl 2-(azidomethyl)morpholine-4-carboxylate

To a stirred solution of tert-butyl2-{[(methanesulfonyl)oxy]methyl}morpholine-4-carboxylate (mixture ofisomers, 2.5 g, 8.46 mmol) in DMF (25 mL), sodium azide (4.64 g, 42.3mmol) and sodium iodide (253 mg, 1.6 mmol) were added at roomtemperature. The reaction mixture was stirred at 60° C. for 20 h thenwas diluted with water (20 mL) and extracted with EtOAc (2×50 mL). Theorganic layer was washed with brine (50 mL), dried over sodium sulfateand concentrated under vacuum. The resulting crude was purified bycolumn chromatography (silica gel: 200-400, 20% EtOAc in pet. ether) toafford the title compound (1.8 g, 87% yield) as a mixture of isomers.LC-MS (ELSD) m/z: 143.2 (M+H-Boc). ¹HNMR (CDCl₃): δ 4.05-3.85 (m, 3H),3.65-3.45 (m, 2H), 3.35-3.00 (m, 2H), 3.00-2.90 (m, 1H), 2.89-2.79 (m,1H), 1.45 (s, 9H).

Step 3—Synthesis of tert-butyl 2-(aminomethyl)morpholine-4-carboxylate

The title intermediate was prepared as a mixture of isomers according tothe procedure described for the synthesis of1-[1-(7-fluoroisoquinolin-1-yl)pyrrolidin-3-yl]methanamine (seecompounds 180-181, step 8) using tert-butyl2-(azidomethyl)morpholine-4-carboxylate (93% yield). LC-MS (ELSD): m/z217.3 (M+H).

Step 4—Synthesis of tert-butyl2-({[(benzyloxy)carbonyl]amino}methyl)morpholine-4-carboxylate

The title intermediate was prepared as a mixture of isomers according tothe procedure described for the synthesis of tert-butyl3-({[(benzyloxy)carbonyl]amino}methyl)pyrrolidine-1-carboxylate (seecompounds 180-181, step 5) using tert-butyl2-(aminomethyl)morpholine-4-carboxylate (54% yield). LC-MS m/z: 251.3(M+H⁺-Boc); ¹HNMR (CDCl₃): δ 7.39-7.33 (m, 5H), 5.13 (s, 2H), 3.88-3.86(m, 3H), 3.73-3.71 (m, 1H), 3.55-3.47 (m, 3H), 3.18-2.88 (m, 1H),2.68-2.65 (m, 1H), 1.48 (s, 9H).

Step 5—Synthesis of benzyl [(morpholin-2-yl)methyl]carbamatehydrochloride

The title intermediate was prepared as a mixture of isomers according tothe procedure described for the synthesis of benzyl[(pyrrolidin-3-yl)methyl]carbamate hydrochloride (see compounds 180-181,step 6) using tert-butyl2-({[(benzyloxy)carbonyl]amino}methyl)morpholine-4-carboxylate (99%yield). LC-MS m/z: 251.1 (M+H⁺).

Step 6—Synthesis of benzyl{[4-(7-fluoroisoquinolin-1-yl)morpholin-2-yl]methyl}carbamate

The title intermediate was prepared as a mixture of isomers according tothe procedure described for the synthesis of benzyl{[1-(7-fluoroisoquinolin-1-yl)pyrrolidin-3-yl]methyl}carbamate (seecompounds 180-181, step 7) using benzyl[(morpholin-2-yl)methyl]carbamate hydrochloride (37% yield). LC-MS m/z:396.2 (M+H⁺).

Step 7—Synthesis of1-[4-(7-fluoroisoquinolin-1-yl)morpholin-2-yl]methanamine

The title intermediate was prepared as a mixture of isomers according tothe procedure described for the synthesis of1-[1-(7-fluoroisoquinolin-1-yl)pyrrolidin-3-yl]methanamine (seecompounds 180-181, step 8) using benzyl{[4-(7-fluoroisoquinolin-1-yl)morpholin-2-yl]methyl}carbamate (84%yield). LC-MS m/z: 262.1 (M+H⁺).

Step 8—Synthesis of the enantiomers of2-[({[4-(7-fluoroisoquinolin-1-yl)morpholin-2-yl]methyl}amino)methyl][1]benzopyrano[4,3-b]pyrrol-4(1H)-one(compounds 182 and 183)

Compounds 182 and 183 were prepared according to the procedure describedfor compounds 180 and 181 (step 9) using the isomeric mixture of1-[4-(7-fluoroisoquinolin-1-yl)morpholin-2-yl]methanamine.

Compound 182 (stereochemistry not assigned): 9.9 mg, 2.6% yield. LC-MSm/z: 459.2 (M+H⁺). ¹H NMR (400 MHz, DMSO-d6): δ 12.35 (brs, 1H), 8.11(d, J=5.6 Hz, 1H), 8.04-7.99 (m, 2H), 7.76 (d, J=10.2 Hz, 1H), 7.66-7.62(m, 1H), 7.47-7.32 (m, 4H), 6.53 (s, 1H), 3.96-3.83 (m, 5H), 3.64-3.48(m, 2H), 3.01 (t, J=10.2 Hz, 1H), 2.82-2.76 (m, 1H), 2.70-2.67 (m, 2H).

Compound 183 (stereochemistry not assigned): 12.9 mg, 3.3% yield. LC-MSm/z: 459.2 (M+H⁺). ¹H NMR (400 MHz, DMSO-d6): δ 12.50 (brs, 1H), 8.12(d, J=5.6 Hz, 1H), 8.05-8.00 (m, 2H), 7.79-7.76 (m, 1H), 7.68-7.63 (m,1H), 7.48-7.34 (m, 4H), 6.57 (s, 1H), 3.97-3.86 (m, 5H), 3.64-3.61 (m,1H), 3.52-3.49 (m, 1H), 3.03-3.00 (m, 1H), 2.83-2.51 (m, 3H).

Preparation of Compound 193

Compound 193 was prepared as described herein below.

Step 1—Synthesis of4-chloro-6-fluoro-2-oxo-2H-1-benzopyran-3-carbaldehyde

POCl₃ (25.8 mL) was added to DMF (40 mL) in one portion at 0° C. Theresulting mixture was heated at 50° C. for 0.5 h then a solution of6-fluoro-4-hydroxycoumarin (10 g, 55.5 mmol) in DMF (30 mL) was added at50° C. The reaction mixture was further heated at 60° C. overnight. Themixture was concentrated in vacuo, treated with toluene (2×50 mL) andevaporated. DCM (400 mL) was added, the resulting mixture was pouredonto ice and stirred for 10 min keeping the temperature around 0° C. Theorganic phase was separated, dried and evaporated in vacuo to obtain12.5 g of the crude product. The title intermediate was progressed intothe next step without further purification and characterization.

Step 2—Synthesis of ethyl8-fluoro-4-oxo-1,4-dihydro[1]benzopyrano[4,3-b]pyrrole-2-carboxylate

4-chloro-6-fluoro-2-oxo-2H-chromene-3-carbaldehyde (12.5 g, crudematerial) and glycine ethyl ester hydrochloride (8.1 g, 58.3 mmol) weresuspended in absolute ethanol (120 mL). TEA (3 eq.) was added at 0° C.then the resulting mixture was stirred at the same temperature for 1hour and at 80° C. for 24 hours. The mixture was concentrated in vacuo,dissolved in DCM (400 mL) and washed with sat. NaHCO₃ (200 mL). Theorganic phase was dried over Na₂SO₄ and evaporated in vacuo. The crudematerial was treated with EtOH (10 mL) to obtain the title compound (7.9g, 28.7 mmol, 51% yield over two steps). LC-MS (M−H⁺)=276.0

Step 3—Synthesis of8-fluoro-2-(hydroxymethyl)[1]benzopyrano[4,3-b]pyrrol-4(1H)-one

8-fluoro-4-oxo-1,4-dihydro[1]benzopyrano[4,3-b]pyrrole-2-carboxylate(7.9 g, 28.7 mmol) was dissolved in dry THF (240 mL). The solution waschilled to −10° C. then LiAlH₄ (1 M solution in THF, 38.2 mL) was slowlyadded. The reaction mixture was stirred at 0° C. for 4 hours then wasquenched by adding Na₂SO₄.10H₂O. The inorganic salts were filtered offand the solvents were evaporated to recover the title product (3.2 g,13.7 mmol, 48% yield), that was progressed without any furtherpurification. LC-MS (M−H⁺)=234.1

Step 4—Synthesis of8-fluoro-4-oxo-1,4-dihydro[1]benzopyrano[4,3-b]pyrrole-2-carbaldehyde

To a solution of8-fluoro-2-(hydroxymethyl)[1]benzopyrano[4,3-b]pyrrol-4(1H)-one (1.2 g,5.15 mmol) in DMSO (12 mL), Dess-Martin periodinane (2.4 g, 5.66 mmol)was added. The mixture was stirred at rt for 30 min then an aqueoussolution of sat. NaHCO₃/10% Na₂S₂O₃ 1:1 was added. The precipitate wasfiltered, washed with water and treated with MeCN (4 mL) and diethylether (10 mL) to obtain the title intermediate (1.1 g, 4.76 mmol, 92%yield). LC-MS (M−H⁺)=232.1

Step 5—Synthesis of 3-amino-5-fluoropyridin-2(1H)-one

5-Fluoro-3-nitropyridin-2-ol (10 g, 63.3 mmol) was dissolved in ethanol(300 mL), 10% Pd/C (1.8 g) was added and the mixture was stirred at roomtemperature under atmospheric pressure of hydrogen for 2 h. Pd/C wasremoved by filtration and the solvent was evaporated in vacuum to obtainthe title compound as an off-white solid (7.5 g, 58.5 mmol, Y=92%).LC-MS (M−H⁺)=129.0

Step 6—Synthesis of tert-butyl{4-[(5-fluoro-2-hydroxypyridin-3-yl)amino]cyclohexyl}carbamate

3-amino-5-fluoropyridin-2(1H)-one (3.7 g, 28.9 mmol) and tert-butyl(4-oxocyclohexyl)carbamate (8 g, 37.6 mmol) were dissolved in DMF (213mL). TFA (18.4 mL, 240 mmol) was added dropwise followed by sodiumtriacetoxyborohydride (9.19 g, 43.4 mmol). The mixture was stirred atroom temperature for 1.5 h then the reaction was quenched with sat.NaHCO₃. The mixture was extracted with ethyl acetate, dried over Na₂SO₄and evaporated under reduced pressure. The residue was purified by flashchromatography (SNAP C-18 400 g, from 100% water+0.1% formic acid to50:50 water+0.1% formic acid/acetonitrile+0.1% formic acid) to give thetitle compound as a formate salt. This material was dissolved in ethylacetate, washed with sat. NaHCO₃ sat. sol., dried over Na₂SO₄ andevaporated under reduced pressure to give the free base of targetcompound as mixture of isomers (5.8 g, 17.8 mmol, 60% yield). LC-MS(M−H⁺)=326.3

Step 7—Synthesis of tert-butyl[4-(7-fluoro-2-oxo-2,3-dihydro-1H-pyrido[2,3-b][1,4]oxazin-1-yl)cyclohexyl]carbamate

To a suspension of tert-butyl{4-[(5-fluoro-2-hydroxypyridin-3-yl)amino]cyclohexyl}carbamate (5.8 g,17.8 mmol) and potassium carbonate (9.9 g, 71.3 mmol) in DMF (110 mL)chloroacetyl chloride (3.1 mL, 39.2 mmol) was added. The mixture wasstirred at room temperature for 30 minutes then was heated to 80° C. for5 h. The mixture was cooled to 0° C. then sat. NaHCO₃ was added (50 mL)followed by ethyl acetate (200 mL). The organic phase was separated,dried over Na₂SO₄ and evaporated in vacuo. The crude product waspurified by Silica gel column (from 100% of cy to cy/ethyl acetate50:50) to obtain the title compound (3.79 g, 10.4 mmol, 58% yield) as amixture of isomers. LC-MS (M−H⁺)=366.4

Step 8—Synthesis of1-(trans-4-aminocyclohexyl)-7-fluoro-1H-pyrido[2,3-b][1,4]oxazin-2(3H)-one

tert-butyl[4-(7-fluoro-2-oxo-2,3-dihydro-1H-pyrido[2,3-b][1,4]oxazin-1-yl)cyclohexyl]carbamate(3.79 g, 10.4 mmol) was dissolved in DCM (70 mL), TFA (10 mL) was addedat 0° C. then the mixture was stirred at rt for 2 h. The solvent wasevaporated in vacuo and the residue was purified by SCX column and thenby preparative HPLC purification under basic conditions (0.1% v/vammonia aqueous solution/acetonitrile) to afford the transdiastereoisomer (1.15 g, 4.3 mmol, 41% yield). LC-MS (M−H⁺)=266.0

Step 9—Synthesis of8-fluoro-2-({[trans-4-(7-fluoro-2-oxo-2,3-dihydro-1H-pyrido[2,3-b][1,4]oxazin-1-yl)cyclohexyl]amino}methyl)[1]benzopyrano[4,3-b]pyrrol-4(1H)-one(hydrochloride, compound 193)

8-fluoro-4-oxo-1,4-dihydro[1]benzopyrano[4,3-b]pyrrole-2-carbaldehyde(158 mg, 0.41 mmol) and1-(trans-4-aminocyclohexyl)-7-fluoro-1H-pyrido[2,3-b][1,4]oxazin-2(3H)-one(100 mg, 0.37 mmol) were suspended in dry dichloromethane (20 mL). 2drops of acetic acid were added. The mixture was stirred for 2 hours at50° C. then NaBH(OAc)₃ (195 mg, 0.925 mmol) was added in one portion.The mixture was stirred at room temperature for 3 h then was partitionedbetween DCM (50 mL) and a sat. NaHCO₃ (20 mL). The organic phase wasdried over Na₂SO₄, filtered and evaporated in vacuo. The crude productwas purified by SCX column, the resulting free base was dissolved inDCM/MeOH (20:1, 12 mL), the mixture was cooled to 0° C. and HCl (1 Msolution in diethyl ether) was added. After stirring at room temperaturefor 10 min the solvent was evaporated in vacuo and the solid was treatedwith diethyl ether and dried to afford 170 mg (0.29 mmol, 78% yield) ofthe title product. LC-MS (M−H⁺)=481.3. ¹H NMR (400 MHz, DMSO-d₆) δ ppm1.55-1.73 (m, 2H), 1.86 (d, J=10.74 Hz, 2H), 2.26 (d, J=11.51 Hz, 2H),2.34-2.47 (m, 2H), 3.24 (br. s., 1H), 4.03-4.17 (m, 1H), 4.37 (br. s.,2H), 4.72 (s, 2H), 6.95 (s, 1H), 7.37 (td, J=8.77, 2.96 Hz, 1H), 7.53(dd, J=9.10, 4.60 Hz, 1H), 7.82-7.90 (m, 2H), 7.97 (dd, J=9.92, 2.47 Hz,1H), 9.33 (br. s., 2H), 13.40 (br. s., 1H).

Preparation of Compound 194

Compound 194 was prepared as described herein below.

Step 1—Synthesis of8-fluoro-4-oxo-1-{[2-(trimethylsilyl)ethoxy]methyl}-1,4-dihydro[1]benzopyrano[4,3-b]pyrrole-2-carbaldehyde

8-fluoro-4-oxo-1,4-dihydro[1]benzopyrano[4,3-b]pyrrole-2-carbaldehyde(see compound 193, 3.5 g, 15.2 mmol) was dissolved in dry THF (120 mL)and cooled to 0° C. TEA (4.2 mL, 30.3 mmol) and SEM-Cl (3.2 mL, 18.2mmol) were added. After stirring 45 min at the same temperature, asaturated solution of NaHCO₃ was added at 0° C. followed by ethylacetate. The organic phase was separated, washed with sat. NH₄Cl andbrine, dried over Na₂SO₄ and evaporated in vacuo. The crude material waspurified by Si-column SNAP 100 eluting with cyclohexane/ethyl acetatefrom 95:5 to 7:3. The resulting residue was treated with diethyl etherand filtered to give the title compound (860 mg, 2.4 mmol, 16% yield).LC-MS (M−H⁺)=362.3

Step 2—Synthesis of8-fluoro-2-[(E)-2-methoxyethenyl]-1-{[2-(trimethylsilyl)ethoxy]methyl}[1]benzopyrano[4,3-b]pyrrol-4(1H)-one

A solution of (methoxymethyl)triphenylphosphonium chloride (1.2 g, 3.6mmol) in THE (20 mL) was cooled to −78° C. LiHMDS (1 M solution in THF,3.6 mL) was added and the mixture was allowed to warm to 0° C. Themixture was cooled again to −78° C. and a solution of8-fluoro-4-oxo-1-{[2-(trimethylsilyl)ethoxy]methyl}-1,4-dihydro[1]benzopyrano[4,3-b]pyrrole-2-carbaldehyde(645 mg, 1.8 mmol) in THF (10 mL) was added dropwise. After stirring for2 hours at −78° C. sat. NH₄Cl was added, the resulting mixture wasdiluted with ethyl acetate and the organic phase was separated, driedand evaporated in vacuo. The crude material was purified by Si-columneluting with cy to cy/ethyl acetate 7:3 to obtain the title intermediate(600 mg, 1.5 mmol, 83% yield) as a mixture of cis/trans isomers. LC-MS(M−H⁺)=390.4

Step 3—Synthesis of(8-fluoro-4-oxo-1,4-dihydro[1]benzopyrano[4,3-b]pyrrol-2-yl)acetaldehyde

TFA (1.5 mL) was added to a solution of8-fluoro-2-[(E)-2-methoxyethenyl]-1-{[2-(trimethylsilyl)ethoxy]methyl}[1]benzopyrano[4,3-b]pyrrol-4(1H)-one(100 mg, 0.26 mmol) in DCM (6 mL). The resulting mixture was stirred at40° C. for 3 hours then was diluted with toluene (60 mL) and evaporatedunder vacuo to afford the title intermediate, that was progressedwithout further purification. LC-MS (M−H⁺)=246.3

Step 4—Synthesis of tert-butyl4-[(5-fluoro-2-hydroxypyridin-3-yl)amino]piperidine-1-carboxylate

The synthesis was performed according to the procedure described for thepreparation of intermediate tert-butyl{4-[(5-fluoro-2-hydroxypyridin-3-yl)amino]cyclohexyl}carbamate (seecompound 193, step 6) using tert-butyl 4-oxopiperidine-1-carboxylate(51% yield). LC-MS (M−H⁺)=312.3

Step 5—Synthesis of tert-butyl4-(7-fluoro-2-oxo-2,3-dihydro-1H-pyrido[2,3-b][1,4]oxazin-1-yl)piperidine-1-carboxylate

The synthesis was performed according to the procedure described for thepreparation of intermediate tert-butyl[4-(7-fluoro-2-oxo-2,3-dihydro-1H-pyrido[2,3-b][1,4]oxazin-1-yl)cyclohexyl]carbamate(see compound 193, step 7) using tert-butyl4-[(5-fluoro-2-hydroxypyridin-3-yl)amino]piperidine-1-carboxylate (46%yield). LC-MS (M−H⁺)=352.3

Step 6—Synthesis of7-fluoro-1-(piperidin-4-yl)-1H-pyrido[2,3-b][1,4]oxazin-2(3H)-one

The synthesis was performed according to the procedure described for thepreparation of intermediate1-(trans-4-aminocyclohexyl)-7-fluoro-1H-pyrido[2,3-b][1,4]oxazin-2(3H)-one(see compound 193, step 8) using tert-butyl4-(7-fluoro-2-oxo-2,3-dihydro-1H-pyrido[2,3-b][1,4]oxazin-1-yl)piperidine-1-carboxylate(87% yield). LC-MS (M−H⁺)=252.3

Step 7—Synthesis of8-fluoro-2-{2-[4-(7-fluoro-2-oxo-2,3-dihydro-1H-pyrido[2,3-b][1,4]oxazin-1-yl)piperidin-1-yl]ethyl}[1]benzopyrano[4,3-b]pyrrol-4(1H)-one(compound 194)

The title compound was prepared according to the procedure described forthe synthesis of8-fluoro-2-({[trans-4-(7-fluoro-2-oxo-2,3-dihydro-1H-pyrido[2,3-b][1,4]oxazin-1-yl)cyclohexyl]amino}methyl)[1]benzopyrano[4,3-b]pyrrol-4(1H)-one(compound 193, step 9) using(8-fluoro-4-oxo-1,4-dihydro[1]benzopyrano[4,3-b]pyrrol-2-yl)acetaldehydeand 7-fluoro-1-(piperidin-4-yl)-1H-pyrido[2,3-b][1,4]oxazin-2(3H)-one(39% yield). LC-MS (M−H⁺)=481.3. ¹H NMR (500 MHz, DMSO-d₆) δ ppm 1.70(d, J=9.74 Hz, 2H), 2.18 (t, J=11.40 Hz, 2H), 2.44-2.61 (m, 2H),2.65-2.73 (m, 2H), 2.90 (t, J=7.48 Hz, 2H), 3.02 (d, J=11.39 Hz, 2H),3.95-4.08 (m, 1H), 4.71 (s, 2H), 6.50 (s, 1H), 7.29 (td, J=8.71, 3.02Hz, 1H), 7.47 (dd, J=8.70, 4.53 Hz, 1H), 7.75-7.93 (m, 3H), 12.39 (br.s., 1H).

Preparation of Compound 197

Compound 197 was prepared as described herein below.

Step 1—Synthesis of tert-butyl4-[(6-methoxy-3-nitropyridin-2-yl)amino]piperidine-1-carboxylate

A mixture of tert-butyl 4-aminopiperidine-1-carboxylate (2.34 g, 11.7mmol), 2-chloro-6-methoxy-3-nitropyridine (3 g, 10.6 mmol) and K₂CO₃(1.47 g, 10.6 mmol) in MeCN (60 mL) and DMF (15 mL) was heated at 60° C.for 3 h. The mixture was filtered and concentrated in vacuo. The residuewas suspended in diethyl ether/water 1:1, the organic phase was driedover MgSO₄ and concentrated. The residue was purified by flashchromatography (silica-gel) to afford the title compound (2.3 g, 6.5mmol, 61% yield). LC-MS (M−H⁺)=353.3

Step 2—Synthesis of tert-butyl4-[(3-amino-6-methoxypyridin-2-yl)amino]piperidine-1-carboxylate

tert-Butyl4-[(6-methoxy-3-nitropyridin-2-yl)amino]piperidine-1-carboxylate (2.3 g,6.5 mmol) was dissolved in ethyl acetate/ethanol 1:1 (60 mL), Pd/C (10%wt, 0.2 g) was added and the mixture was stirred at room temperatureunder atmospheric pressure of hydrogen for 24 h. Pd/C was removed byfiltration and the solvent was evaporated in vacuo. The resultingresidue was purified by Si-column eluting with cy to cy/ethyl acetate1:1 to obtain the title product (1.2 g, 3.7 mmol, 57% yield). LC-MS(M−H⁺)=323.4

Step 3—Synthesis of tert-butyl4-({3-[(2-ethoxy-2-oxoethyl)amino]-6-methoxypyridin-2-yl}amino)piperidine-1-carboxylate

tert-Butyl4-[(3-amino-6-methoxypyridin-2-yl)amino]piperidine-1-carboxylate (1.2 g,3.7 mmol) was dissolved in DMF/MeCN 1:2 (30 mL), potassium carbonate(513 mg, 3.7 mmol) was added followed by ethyl bromoacetate (0.43 mL,3.8 mmol). The mixture was stirred at room temperature overnight thenethyl acetate was added followed by water. The organic phase wasseparated, washed with brine, dried and evaporated in vacuo. The crudematerial was purified by Si-column eluting with cy to cy/ethyl acetate7:3 to obtain the title intermediate (1.4 g, 3.4 mmol, 92% yield). LC-MS(M−H⁺)=409.5

Step 4—Synthesis of tert-butyl4-(6-methoxy-3-oxo-2,3-dihydropyrido[2,3-b]pyrazin-4(1H)-yl)piperidine-1-carboxylate

A solution of tert-butyl4-({3-[(2-ethoxy-2-oxoethyl)amino]-6-methoxypyridin-2-yl}amino)piperidine-1-carboxylate(500 mg, 1.2 mmol) and cat. acetic acid (0.1 mL) in toluene (25 mL) wasrefluxed overnight. The reaction mixture was concentrated, the residuewas dissolved in DCM (20 mL) and treated with MnO₂ (2 g) at roomtemperature for 2 hours. The solid was filtered and the solvent wasevaporated in vacuo. The crude material was purified by Si-columneluting with cy to cy/ethyl acetate 8:2 to obtain the title compound(137 mg, 0.38 mmol, 32% yield). LC-MS (M-Na⁺)=383.3

Step 5—Synthesis of6-methoxy-4-(piperidin-4-yl)pyrido[2,3-b]pyrazin-3(4H)-one

The synthesis was performed according to the procedure described for thepreparation of intermediate1-(trans-4-aminocyclohexyl)-7-fluoro-1H-pyrido[2,3-b][1,4]oxazin-2(3H)-one(see compound 193, step 8) using tert-butyl4-(6-methoxy-3-oxo-2,3-dihydropyrido[2,3-b]pyrazin-4(1H)-yl)piperidine-1-carboxylate(98% yield). LC-MS (M−H⁺)=261.3

Step 6—Synthesis of8-fluoro-2-{2-[4-(6-methoxy-3-oxopyrido[2,3-b]pyrazin-4(3H)-yl)piperidin-1-yl]ethyl}[1]benzopyrano[4,3-b]pyrrol-4(1H)-one

The title compound was prepared according to the procedure described forthe synthesis of8-fluoro-2-({[trans-4-(7-fluoro-2-oxo-2,3-dihydro-1H-pyrido[2,3-b][1,4]oxazin-1-yl)cyclohexyl]amino}methyl)[1]benzopyrano[4,3-b]pyrrol-4(1H)-one(compound 193, step 9) using(8-fluoro-4-oxo-1,4-dihydro[1]benzopyrano[4,3-b]pyrrol-2-yl)acetaldehydeand 6-methoxy-4-(piperidin-4-yl)pyrido[2,3-b]pyrazin-3(4H)-one (11%yield). LC-MS (M−H⁺)=490.5. ¹H NMR (500 MHz, DMSO-d₆) δ ppm 1.62 (d,J=10.43 Hz, 2H), 2.13 (t, J=11.05 Hz, 2H), 2.66-2.77 (m, 2H), 2.85-3.01(m, 4H), 3.12 (d, J=11.39 Hz, 2H), 3.92 (s, 3H), 5.25 (br. s., 1H), 6.52(s, 1H), 6.84 (d, J=8.64 Hz, 1H), 7.29 (td, J=8.71, 3.02 Hz, 1H), 7.48(dd, J=9.06, 4.53 Hz, 1H), 7.81 (dd, J=8.92, 3.02 Hz, 1H), 8.07 (s, 1H),8.12 (d, J=8.64 Hz, 1H), 12.40 (br. s., 1H).

Preparation of Compound 200

Compound 200 was prepared as described herein below.

Step 1—Synthesis of 3-chloro-6-methoxypyrazin-2-amine

To a solution of 6-methoxypyrazin-2-amine (3 g, 24 mmol) in dry DMF (35mL), N-chlorosuccinimide (3.2 g, 24 mmol) was added. The reactionmixture was stirred at room temperature for 16 h then was poured intoice and brine (130 mL). The aqueous solution was extracted with ethylacetate (3×120 mL), the organic phase was washed with 5% solution ofLiCl, dried over Na₂SO₄ and evaporated under reduced pressure. The crudewas purified by SNAP-340-NH (cyclohexane/ethyl acetate from 95:5 up to8:2) and by SNAP100-Si—OH (eluting with DCM) to afford the titleintermediate (1.98 g, 12.5 mmol, 52% yield). LC-MS (M−H⁺)=160.1

Step 2—Synthesis of tert-butyl4-[(3-chloro-6-methoxypyrazin-2-yl)amino]piperidine-1-carboxylate

A solution of titanium (IV) chloride (1 M in dichloromethane, 5.8 mL)was added dropwise at 0° C. to a stirred solution of3-chloro-6-methoxypyrazin-2-amine (1.85 g, 11.6 mmol), tert-butyl4-oxopiperidine-1-carboxylate (2.55 g, 12.8 mmol) and triethylamine(4.85 mL, 34.8 mmol) in dry dichloromethane (58 mL). The reactionmixture was slowly allowed to warm to room temperature and stirredovernight. To the resulting mixture a solution of sodiumcyanoborohydride (2.18 g, 34.8 mmol) in MeOH (23 mL) was then added atroom temperature. After 4 hours the reaction mixture was quenched with a3 M NaOH solution (180 mL). EtOAc (600 mL) was then added and themixture was filtered onto a Celite pad (10 cm diameter, 3 cm height).The phases were separated and the organic layer was washed with aqueoussodium bicarbonate solution and brine, dried over sodium sulfate andevaporated under reduced pressure. The crude was purified by flashchromatography (Biotage KP-Sil 100 g SNAP cartridge, eluent from 100%DCM to DCM/MeOH 95:5) to give the title compound (1 g, 2.9 mmol, 25%yield). LC-MS (M−H⁺)=343.4

Step 3—Synthesis of tert-butyl4-({3-[(1E)-3-ethoxy-3-oxoprop-1-en-1-yl]-6-methoxypyrazin-2-yl}amino)piperidine-1-carboxylate

tert-Butyl4-[(3-chloro-6-methoxypyrazin-2-yl)amino]piperidine-1-carboxylate (0.764g, 2.23 mmol), Pd(PtBu₃)₃(0.08 g, 0.156 mmol, 7% mol) and ethyl acrylate(0.29 mL, 2.67 mmol) were suspended in triethylamine (5.0 mL). Theresulting mixture was stirred for 90 min at 130° C. then was cooled andpartitioned between water and EtOAc. The aqueous layer was extractedtwice with EtOAc, the combined organic layers were washed with brine,dried over Na₂SO₄ and concentrated under vacuum. The resulting crude waspurified by column chromatography on SiO₂ (cyclohexane/EtOAc from 9:1 to1:1) to afford the title compound (448 mg, 1.1 mmol, 49% yield). LC-MS(M−H⁺)=407.5

Step 4—Synthesis of tert-butyl4-(3-methoxy-6-oxopyrido[2,3-b]pyrazin-5(6H)-yl)piperidine-1-carboxylate

Intermediate tert-butyl4-({3-[(1E)-3-ethoxy-3-oxoprop-1-en-1-yl]-6-methoxypyrazin-2-yl}amino)piperidine-1-carboxylate(0.34 g, 0.85 mmol) was dissolved in MeOH (17 mL), potassium carbonate(234 mg, 1.69 mmol) was added and the mixture was stirred at 65° C. for24 h. The volatiles were removed under vacuum, the residue waspartitioned between EtOAc and H₂O and the aqueous layer was extractedwith EtOAc. The combined organic layers were washed with brine and driedover Na₂SO₄. The solvent was removed under vacuum to afford a crudewhich was purified by column chromatography (from cyclohexane/EtOAc 7:3to EtOAc) to give the title compound (177 mg, 0.49 mmol, 58% yield).LC-MS (M−H⁺)=361.4

Step 5—Synthesis of3-methoxy-5-(piperidin-4-yl)pyrido[2,3-b]pyrazin-6(5H)-one

The synthesis was performed according to the procedure described for thepreparation of intermediate1-(trans-4-aminocyclohexyl)-7-fluoro-1H-pyrido[2,3-b][1,4]oxazin-2(3H)-one(see compound 193, step 8) using tert-butyl4-(3-methoxy-6-oxopyrido[2,3-b]pyrazin-5(6H)-yl)piperidine-1-carboxylate(92% yield). LC-MS (M−H⁺)=261.3

Step 6—Synthesis of8-fluoro-2-{2-[4-(6-methoxy-3-oxopyrido[2,3-b]pyrazin-4(3H)-yl)piperidin-1-yl]ethyl}[1]benzopyrano[4,3-b]pyrrol-4(1H)-one(hydrochloride, compound 200)

The title compound was prepared according to the procedure described forthe synthesis of8-fluoro-2-({[trans-4-(7-fluoro-2-oxo-2,3-dihydro-1H-pyrido[2,3-b][1,4]oxazin-1-yl)cyclohexyl]amino}methyl)[1]benzopyrano[4,3-b]pyrrol-4(1H)-one(compound 193, step 9) using(8-fluoro-4-oxo-1,4-dihydro[1]benzopyrano[4,3-b]pyrrol-2-yl)acetaldehydeand 3-methoxy-5-(piperidin-4-yl)pyrido[2,3-b]pyrazin-6(5H)-one (12%yield). LC-MS (M−H⁺)=490.4. ¹H NMR (500 MHz, METHANOL-d₄) δ ppm 2.08 (d,J=13.69 Hz, 2H), 3.32-3.43 (m, 6H), 3.55-3.66 (m, 2H), 3.87 (d, J=10.76Hz, 2H), 4.18 (s, 3H), 5.87 (br. s., 1H), 6.64-6.78 (m, 2H), 7.24 (td,J=8.56, 2.93 Hz, 1H), 7.45 (dd, J=9.29, 4.40 Hz, 1H), 7.64 (dd, J=8.31,2.93 Hz, 1H), 7.96 (d, J=9.29 Hz, 1H), 8.21 (s, 1H).

Preparation of Compound 201

Compound 201 was prepared as described herein below.

Step 1—Synthesis of tert-butyl{trans-4-[(6-methoxy-3-nitropyridin-2-yl)amino]cyclohexyl}carbamate

To a solution of tert-butyl (trans-4-aminocyclohexyl)carbamate (1.7 g, 8mmol) and TEA (1.1 mL, 8 mmol) in acetonitrile (53 mL)2-chloro-6-methoxy-3-nitropyridine (1.5 g, 7.95 mmol) was added. Thereaction mixture was heated at 90° C. overnight then was cooled,filtered, and the filtrate was concentrated. The residue was treatedwith 50 mL of hot ethyl acetate, filtered and concentrated. The residuewas dissolved in DCM and washed with sat. NH₄Cl. The organic phase wasdried over Na₂SO₄ and concentrated to afford the title compound (1.5 g,6.7 mmol, 84% yield). LC-MS (M−H⁺)=367.4

Step 2—Synthesis of tert-butyl{trans-4-[(3-amino-6-methoxypyridin-2-yl)amino]cyclohexyl}carbamate

The title compound was prepared according to the procedure described forthe synthesis of tert-butyl4-[(3-amino-6-methoxypyridin-2-yl)amino]piperidine-1-carboxylate (seecompound 197, step 2) using tert-butyl{trans-4-[(6-methoxy-3-nitropyridin-2-yl)amino]cyclohexyl}carbamate (94%yield). LC-MS (M−H⁺)=337.4

Step 3—Synthesis of ethylN-[2-({trans-4-[(tert-butoxycarbonyl)amino]cyclohexyl}amino)-6-methoxypyridin-3-yl]glycinate

The title compound was prepared according to the procedure described forthe synthesis of tert-butyl4-({3-[(2-ethoxy-2-oxoethyl)amino]-6-methoxypyridin-2-yl}amino)piperidine-1-carboxylate(see compound 197, step 3) using tert-butyl{trans-4-[(3-amino-6-methoxypyridin-2-yl)amino]cyclohexyl}carbamate (62%yield). LC-MS (M−H⁺)=423.5

Step 4—Synthesis of tert-butyl[trans-4-(6-methoxy-3-oxopyrido[2,3-b]pyrazin-4(3H)-yl)cyclohexyl]carbamate

The title compound was prepared according to the procedure described forthe synthesis of tert-butyl4-(6-methoxy-3-oxo-2,3-dihydropyrido[2,3-b]pyrazin-4(1H)-yl)piperidine-1-carboxylate(see compound 197, step 4) using ethylN-[2-({trans-4-[(tert-butoxycarbonyl)amino]cyclohexyl}amino)-6-methoxypyridin-3-yl]glycinate(51% yield). LC-MS (M-Na⁺)=397.5

Step 5—Synthesis of4-(trans-4-aminocyclohexyl)-6-methoxypyrido[2,3-b]pyrazin-3(4H)-one

The title compound was prepared according to the procedure described forthe synthesis of1-(trans-4-aminocyclohexyl)-7-fluoro-1H-pyrido[2,3-b][1,4]oxazin-2(3H)-one(see compound 193, step 8) using tert-butyl[trans-4-(6-methoxy-3-oxopyrido[2,3-b]pyrazin-4(3H)-yl)cyclohexyl]carbamate(87% yield). LC-MS (M-Na⁺)=275.3

Step 6—Synthesis of8-fluoro-2-({[trans-4-(6-methoxy-3-oxopyrido[2,3-b]pyrazin-4(3H)-yl)cyclohexyl]amino}methyl)[1]benzopyrano[4,3-b]pyrrol-4(1H)-one(formate salt, compound 201)

The title compound was prepared according to the procedure described forthe synthesis of8-fluoro-2-({[trans-4-(7-fluoro-2-oxo-2,3-dihydro-1H-pyrido[2,3-b][1,4]oxazin-1-yl)cyclohexyl]amino}methyl)[1]benzopyrano[4,3-b]pyrrol-4(1H)-one(compound 193, step 9) using4-(trans-4-aminocyclohexyl)-6-methoxypyrido[2,3-b]pyrazin-3(4H)-one (17%yield). LC-MS (M−H⁺)=490.3. ¹H NMR (500 MHz, DMSO-d₆) δ ppm 1.21-1.32(m, 2H), 1.67 (d, J=10.27 Hz, 2H), 2.10 (d, J=11.98 Hz, 2H), 2.41-2.56(m, 1H), 2.72 (br. s., 2H), 3.83-3.96 (m, 5H), 4.96-5.49 (m, 1H), 6.58(s, 1H), 6.83 (d, J=8.56 Hz, 1H), 7.29 (td, J=8.80, 2.93 Hz, 1H), 7.48(dd, J=9.05, 4.65 Hz, 1H), 7.95 (dd, J=9.05, 2.93 Hz, 1H), 8.00-8.07 (m,1H), 8.10 (d, J=8.56 Hz, 1H), 8.22 (s, 1H), 12.61 (br. s, 2H).

Preparation of Compound 202

Compound 202 was prepared as described herein below.

Step 1—Synthesis of N-(6-chloropyridin-2-yl)-2,2-dimethylpropanamide

To a solution of 2-amino-6-chloropyridine (10.7 g, 83.5 mmol) in toluene(103 mL) NaHCO₃ (14 g, 167 mmol) and pivaloyl chloride (15.4 mL, 125.2mmol) were added at 0° C. The resulting mixture was stirred at roomtemperature for 5 hours then the suspension was filtered and the solidwas washed with DCM. The filtrates were concentrated under vacuum thenheptane (22 mL) was added and the resulting mixture was concentrated.The solid was filtered, washed with heptane (15 mL) and dried undervacuum to afford the title intermediate (15.4 g, 72.4 mmol, 87% yield).LC-MS (M−H⁺)=213.2

Step 2—Synthesis ofN-(6-chloro-3-formylpyridin-2-yl)-2,2-dimethylpropanamide

To a solution of N-(6-chloropyridin-2-yl)-2,2-dimethylpropanamide (10.6g, 50 mmol) in THF (100 mL) n-butyllithium (2.5 M solution in hexane, 50mL, 125 mmol) was added. The resulting mixture was stirred at −20° C.for 3 h. After the addiction of DMF (4 mL) the reaction mixture wasallowed to warm to room temperature and then was quenched with 0.5 MHCl. Ethyl acetate was added, the organic phase was washed with water,sat. K₂CO₃ and with brine. The crude material was purified by Si-columneluting with cy to cy/ethyl acetate 7:3 to obtain the title product (6.7g, 27.8 mmol, 56% yield). LC-MS (M−H⁺)=241.3

Step 3—Synthesis of tert-butyl3-[6-chloro-2-(2,2-dimethylpropanamido)pyridin-3-yl]-3-hydroxypropanoate

An oven-dried flask was charged with THF (80 mL) and diisopropylamine(8.2 mL, 58.4 mmol). The solution was cooled to −78° C. thenn-butyllithium (2.5 M solution in hexane, 23.3 mL, 58.3 mmol) was added.The mixture was stirred for 15 min then a solution of tert-butyl acetate(7.8 mL, 58.3 mmol) in THF (2 mL) was added. After stirring at −78° C.for 20 min a solution ofN-(6-chloro-3-formylpyridin-2-yl)-2,2-dimethylpropanamide (6.7 g, 27.8mmol) in THF (5 mL) was added. The mixture was allowed to warm to roomtemperature then was poured into sat. NH₄Cl. The resulting mixture wasextracted with ethyl acetate and the organic phase was dried andevaporated in vacuo to obtain the title compound (9.4 g, 26.4 mmol, 95%yield). LC-MS (M−H⁺)=357.3

Step 4—Synthesis of 7-chloro-1,8-naphthyridin-2(1H)-one

Intermediate tert-butyl3-[6-chloro-2-(2,2-dimethylpropanamido)pyridin-3-yl]-3-hydroxypropanoate(4 g, 11 mmol) was dissolved in 1 M HCl/dioxane 1:1 (50 mL) and refluxedovernight. Ice was added to the mixture and the resulting solid wasfiltered and dried to afford the title product (1.7 g, 9.4 mmol, 86%yield), that was used without any further purification. LC-MS(M−H⁺)=181.1

Step 5—Synthesis of 7-methoxy-1,8-naphthyridin-2(1H)-one

To a suspension of 7-chloro-1,8-naphthyridin-2(1H)-one (700 mg, 3.9mmol) in MeOH (15 mL), NaOMe (25% solution in MeOH, 20 mL) was added.The resulting solution was stirred at reflux for 15 h then the solventwas removed in vacuo. Water (100 mL) and EtOAc (80 mL) were added, thephases were separated and the aqueous layer was extracted with EtOAc(8×80 mL). The combined organic layers were washed with brine (50 mL),dried over MgSO₄, filtered and concentrated under reduced pressure togive the title compound (630 mg, 3.6 mmol, 92% yield). LC-MS(M−H⁺)=177.2

Step 6—Synthesis of tert-butyl4-(7-methoxy-2-oxo-1,8-naphthyridin-1(2H)-yl)piperidine-1-carboxylate

Cs₂CO₃ (828 mg, 2.6 mmol) and tert-butyl 4-bromopiperidine-1-carboxylate(1.35 g, 5.1 mmol) were sequentially added to a solution of7-methoxy-1,8-naphthyridin-2(1H)-one (150 mg, 0.85 mmol) in DMF (15 mL).The reaction mixture was stirred at 50° C. overnight then waspartitioned between EtOAc and water. The organic phases were dried overNa₂SO₄, filtered and evaporated in vacuo. The crude material waspurified by silica-gel chromatography (cyclohexane/EtOAc from 100:0 to50:50) to afford the title product (70 mg, 0.2 mmol, 22% yield). LC-MS(M−H⁺)=360.4

Step 7—Synthesis of7-methoxy-1-(piperidin-4-yl)-1,8-naphthyridin-2(1H)-one

The title intermediate was prepared according to the procedure describedfor the synthesis of1-(trans-4-aminocyclohexyl)-7-fluoro-1H-pyrido[2,3-b][1,4]oxazin-2(3H)-one(see compound 193, step 8) using tert-butyl4-(7-methoxy-2-oxo-1,8-naphthyridin-1(2H)-yl)piperidine-1-carboxylate(72% yield). LC-MS (M−H⁺)=260.3

Step 8—Synthesis of8-fluoro-2-{2-[4-(7-methoxy-2-oxo-1,8-naphthyridin-1(2H)-yl)piperidin-1-yl]ethyl}[1]benzopyrano[4,3-b]pyrrol-4(1H)-one(hydrochloride, compound 202)

The title compound was prepared according to the procedure described forthe synthesis of8-fluoro-2-({[trans-4-(7-fluoro-2-oxo-2,3-dihydro-1H-pyrido[2,3-b][1,4]oxazin-1-yl)cyclohexyl]amino}methyl)[1]benzopyrano[4,3-b]pyrrol-4(1H)-one(compound 193, step 9) using(8-fluoro-4-oxo-1,4-dihydro[1]benzopyrano[4,3-b]pyrrol-2-yl)acetaldehydeand 7-methoxy-1-(piperidin-4-yl)-1,8-naphthyridin-2(1H)-one (11% yield).LC-MS (M−H⁺)=489.3. ¹H NMR (500 MHz, METHANOL-d₄) δ ppm 1.95-2.11 (m,2H), 3.30-3.40 (m, 6H), 3.55-3.63 (m, 2H), 3.81-3.88 (m, 2H), 4.08 (s,3H), 6.03 (br. s., 1H), 6.50 (d, J=9.39 Hz, 1H), 6.69 (s, 1H), 6.75 (d,J=8.41 Hz, 1H), 7.22 (td, J=8.71, 2.93 Hz, 1H), 7.43 (dd, J=9.10, 4.40Hz, 1H), 7.62 (dd, J=8.61, 2.93 Hz, 1H), 7.83 (d, J=9.39 Hz, 1H), 7.98(d, J=8.61 Hz, 1H).

Preparation of Compounds 204 and 205

Compounds 204 and 205 were prepared as described herein below.

Step 1—Synthesis of tert-butyl{4-[(2-bromo-5-fluoropyridin-3-yl)amino]cyclohexyl}carbamate

The title compound was obtained as a mixture of isomers following theprocedure described for the preparation of intermediate tert-butyl{4-[(5-fluoro-2-hydroxypyridin-3-yl)amino]cyclohexyl}carbamate (seecompound 193, step 6) using 2-bromo-5-fluoropyridin-3-amine (98% yield).LC-MS (M−H⁺)=388.2

Step 2—Synthesis of ethyl(2E)-3-[3-({4-[(tert-butoxycarbonyl)amino]cyclohexyl}amino)-5-fluoropyridin-2-yl]prop-2-enoate

The title compound was obtained as a mixture of isomers following theprocedure described for the preparation of intermediate tert-butyl4-({3-[(1E)-3-ethoxy-3-oxoprop-1-en-1-yl]-6-methoxypyrazin-2-yl}amino)piperidine-1-carboxylate(see compound 200, step 3) using tert-butyl{4-[(2-bromo-5-fluoropyridin-3-yl)amino]cyclohexyl}carbamate (66%yield). LC-MS (M−H⁺)=408.5

Step 3—Synthesis of tert-butyl4-({3-[(1E)-3-ethoxy-3-oxoprop-1-en-1-yl]-6-methoxypyrazin-2-yl}amino)piperidine-1-carboxylate

The title compound was obtained as a mixture of isomers following theprocedure described for the preparation of intermediate tert-butyl4-(3-methoxy-6-oxopyrido[2,3-b]pyrazin-5(6H)-yl)piperidine-1-carboxylate(see compound 200, step 4) using ethyl(2E)-3-[3-({4-[(tert-butoxycarbonyl)amino]cyclohexyl}amino)-5-fluoropyridin-2-yl]prop-2-enoate(17% yield). LC-MS (M−H⁺)=374.3

Step 4—Synthesis of1-(4-aminocyclohexyl)-7-methoxy-1,5-naphthyridin-2(1H)-one

The title compound was obtained as a mixture of isomers following theprocedure described for the preparation of intermediate1-(trans-4-aminocyclohexyl)-7-fluoro-1H-pyrido[2,3-b][1,4]oxazin-2(3H)-one(see compound 193, step 8) using tert-butyl4-({3-[(1E)-3-ethoxy-3-oxoprop-1-en-1-yl]-6-methoxypyrazin-2-yl}amino)piperidine-1-carboxylate(73% yield). LC-MS (M−H⁺)=274.1

Step 5—Synthesis of8-fluoro-2-({[trans-4-(7-methoxy-2-oxo-1,5-naphthyridin-1(2H)-yl)cyclohexyl]amino}methyl)[1]benzopyrano[4,3-b]pyrrol-4(1H)-one(hydrochloride, compound 204) and8-fluoro-2-({[cis-4-(7-methoxy-2-oxo-1,5-naphthyridin-1(2H)-yl)cyclohexyl]amino}methyl)[1]benzopyrano[4,3-b]pyrrol-4(1H)-one(hydrochloride, compound 205)

The title compounds were prepared according to the procedure describedfor the synthesis of8-fluoro-2-({[trans-4-(7-fluoro-2-oxo-2,3-dihydro-1H-pyrido[2,3-b][1,4]oxazin-1-yl)cyclohexyl]amino}methyl)[1]benzopyrano[4,3-b]pyrrol-4(1H)-one(compound 193, step 9) using1-(4-aminocyclohexyl)-7-methoxy-1,5-naphthyridin-2(1H)-one as a mixtureof isomers.

A purification by preparative HPLC (A=0.1% ammonia aqueous solution,B=acetonitrile, form 98:2 A:B to 100% B) followed by treatment with HClunder the previously described conditions afforded the trans (20% yield)and cis (15% yield) diastereoisomers.

Compound 204: LC-MS (M−H⁺)=489.2; ¹H NMR (400 MHz, DMSO-d₆) δ ppm1.62-1.91 (m, 4H), 2.20-2.40 (m, 2H), 2.67 (br. s., 2H), 3.28 (br. s.,1H), 4.01 (s, 3H), 4.34-4.86 (m, 3H), 6.60 (d, J=9.59 Hz, 1H), 6.95 (d,J=1.57 Hz, 1H), 7.38 (td, J=8.75, 3.03 Hz, 1H), 7.53 (dd, J=9.10, 4.50Hz, 1H), 7.63 (br. s., 1H), 7.78-7.92 (m, 2H), 8.32 (d, J=2.15 Hz, 1H),9.33 (br. s., 2H), 13.41 (br. s., 1H).

Compound 205: LC-MS (M−H⁺)=489.2; ¹H NMR (400 MHz, DMSO-d₆) δ ppm 1.65(d, J=11.74 Hz, 2H), 2.00 (m, J=13.89 Hz, 2H), 2.17 (d, J=14.28 Hz, 2H),2.76 (m, J=12.13 Hz, 2H), 3.40-3.56 (m, 1H), 4.01 (s, 3H), 4.39-4.51 (m,2H), 4.59-4.92 (m, 1H), 6.62 (d, J=9.59 Hz, 1H), 6.99 (d, J=1.76 Hz,1H), 7.38 (td, J=8.75, 3.03 Hz, 1H), 7.53 (dd, J=9.10, 4.60 Hz, 1H),7.67 (s, 1H), 7.78-7.92 (m, 2H), 8.33 (d, J=2.15 Hz, 1H), 9.23 (br. s.,2H), 13.49 (s, 1H).

Preparation of Compound 206

Compound 206 was prepared as described herein below.

Step 1—Synthesis of tert-butyl[4-(5-cyano-2-iodoanilino)cyclohexyl]carbamate

The title compound was obtained as a mixture of isomers following theprocedure described for the preparation of intermediate tert-butyl{4-[(5-fluoro-2-hydroxypyridin-3-yl)amino]cyclohexyl}carbamate (seecompound 193, step 6) using 3-amino-4-iodobenzonitrile (24% yield).LC-MS (M−H⁺)=442.2

Step 2—Synthesis of ethyl(2E)-3-[2-({4-[(tert-butoxycarbonyl)amino]cyclohexyl}amino)-4-cyanophenyl]prop-2-enoate

The title compound was obtained as a mixture of isomers following theprocedure described for the preparation of intermediate tert-butyl4-({3-[(1E)-3-ethoxy-3-oxoprop-1-en-1-yl]-6-methoxypyrazin-2-yl}amino)piperidine-1-carboxylate(see compound 200, step 3) using tert-butyl[4-(5-cyano-2-iodoanilino)cyclohexyl]carbamate (88% yield). LC-MS(M−H⁺)=414.4

Step 3—Synthesis of ethyl3-[2-({4-[(tert-butoxycarbonyl)amino]cyclohexyl}amino)-4-cyanophenyl]propanoate

Ethyl(2E)-3-{2-[(4-{[(tert-butoxy)carbonyl]amino}cyclohexyl)amino]-4-cyanophenyl}prop-2-enoate(37.9 g, 91.7 mmol) was dissolved in ethyl acetate (379 mL), 10% Pd/C(7.6 g) was added and the mixture was stirred under hydrogen atmosphere(1 atm) overnight. The catalyst was removed by filtration and thesolvent was evaporated in vacuo to obtain the title intermediate as amixture of isomers (35.7 g, 85.9 mmol, Y=94%, LC-MS (M−H⁺)=416.4

Step 4—Synthesis of3-[2-({4-[(tert-butoxycarbonyl)amino]cyclohexyl}amino)-4-cyanophenyl]propanoic

Ethyl3-{2-[(4-{[(tert-butoxy)carbonyl]amino}cyclohexyl)amino]-4-cyanophenyl}propanoate(43.5 g, 0.11 mol) was dissolved in THF/H₂O (455/136 mL), LiOH.H₂O (8.8g, 0.21 mol) was added and the mixture was stirred at room temperatureovernight. The solvent was evaporated in vacuo, the residue was treatedwith acetonitrile, filtered and concentrated in vacuo. The crude titleproduct (mixture of isomers) was progressed without any furtherpurification.

Step 5—Synthesis of tert-butyl[trans-4-(7-cyano-2-oxo-3,4-dihydroquinolin-1(2H)-yl)cyclohexyl]carbamate

3-[2-({4-[(tert-butoxycarbonyl)amino]cyclohexyl}amino)-4-cyanophenyl]propanoicacid (0.11 mol from previous step) was dissolved in DMF (910 mL), TEA(30.5 mL, 0.22 mol) was added and the mixture was cooled to 0° C. HATU(41.6 g, 0.11 mol) was added at the same temperature and the mixture wasallowed to slowly reach room temperature. After stirring overnight themixture was partitioned between water (1 L) and EtOAc (500 mL). Theorganic phase was washed with brine and dried over Na₂SO₄. The volatileswere removed under vacuum and the crude residue was purified bySi-column (cyclohexane/ethyl acetate from 8:2 to 1:1) to obtain thetitle trans diastereoisomer (14.4 g, 39 mmol, 35% yield over two steps).LC-MS (M−H⁺)=370.2

Step 6—Synthesis of tert-butyl[trans-4-(7-cyano-2-oxoquinolin-1(2H)-yl)cyclohexyl]carbamate

tert-Butyl[trans-4-(7-cyano-2-oxo-3,4-dihydroquinolin-1(2H)-yl)cyclohexyl]carbamate(13.7 g 37.2 mmol) was dissolved in DCE (274 mL), N-bromosuccinimide(8.6 g, 48.4 mmol) and 2,2′-azobis(2-methylpropionitrile) (917 mg, 5.6mmol) were added and the mixture was stirred at 80° C. for 4 h. Water(100 mL) was added, the organic phase was separated, washed with brine(80 mL) and evaporated in vacuo to obtain the crude title product thatwas progressed without any further purification. LC-MS (M−H⁺)=368.3

Step 7—Synthesis of1-(trans-4-aminocyclohexyl)-2-oxo-1,2-dihydroquinoline-7-carbonitrile

The title intermediate was prepared according to the procedure describedfor the synthesis of1-(trans-4-aminocyclohexyl)-7-fluoro-1H-pyrido[2,3-b][1,4]oxazin-2(3H)-one(see compound 193, step 8) using tert-butyl[trans-4-(7-cyano-2-oxoquinolin-1(2H)-yl)cyclohexyl]carbamate (quant.yield). LC-MS (M−H⁺)=268.2

Step 8—Synthesis of1-(trans-4-{[(8-fluoro-4-oxo-1,4-dihydro[1]benzopyrano[4,3-b]pyrrol-2-yl)methyl]amino}cyclohexyl)-2-oxo-1,2-dihydroquinoline-7-carbonitrile(hydrochloride, compound 206)

The title compound was prepared according to the procedure described forthe synthesis of8-fluoro-2-({[trans-4-(7-fluoro-2-oxo-2,3-dihydro-1H-pyrido[2,3-b][1,4]oxazin-1-yl)cyclohexyl]amino}methyl)[1]benzopyrano[4,3-b]pyrrol-4(1H)-one(compound 193, step 9) using1-(trans-4-aminocyclohexyl)-2-oxo-1,2-dihydroquinoline-7-carbonitrile(12% yield). LC-MS (M−H⁺)=483.3. ¹H NMR (500 MHz, DMSO-d₆) δ ppm1.66-1.90 (m, 4H), 2.19-2.40 (m, 2H), 2.61-2.82 (m, 2H), 3.08-3.42 (m,1H), 4.40 (br. s., 2H), 4.64 (br. s., 1H), 6.70 (d, J=8.51 Hz, 1H), 6.96(br. s., 1H), 7.37 (td, J=8.71, 2.88 Hz, 1H), 7.53 (dd, J=9.13, 4.60 Hz,1H), 7.66 (d, J=8.10 Hz, 1H), 7.85-7.93 (m, 2H), 7.96 (d, J=9.61 Hz,1H), 8.40 (br. s., 1H), 9.44 (br. s., 1H), 13.53 (br. s., 1H).

Preparation of Compound 207

Compound 207 was prepared as described herein below.

Step 1—Synthesis of tert-butyl{4-[(2-bromo-5-fluoropyridin-3-yl)amino]cyclohexyl}carbamate

The title compound was obtained as a mixture of isomers following theprocedure described for the preparation of intermediate tert-butyl{4-[(5-fluoro-2-hydroxypyridin-3-yl)amino]cyclohexyl}carbamate (seecompound 193, step 6) using 2-bromo-5-fluoropyridin-3-amine (98% yield).LC-MS (M−H⁺)=388.2

Step 2—Synthesis of ethyl(2E)-3-[3-({4-[(tert-butoxycarbonyl)amino]cyclohexyl}amino)-5-fluoropyridin-2-yl]prop-2-enoate

The title compound was obtained as a mixture of isomers following theprocedure described for the preparation of intermediate tert-butyl4-({3-[(1E)-3-ethoxy-3-oxoprop-1-en-1-yl]-6-methoxypyrazin-2-yl}amino)piperidine-1-carboxylate(see compound 200, step 3) using tert-butyl{4-[(2-bromo-5-fluoropyridin-3-yl)amino]cyclohexyl}carbamate (66%yield). LC-MS (M−H⁺)=408.5

Step 3—Synthesis of ethyl3-[3-({4-[(tert-butoxycarbonyl)amino]cyclohexyl}amino)-5-fluoropyridin-2-yl]propanoate

The title compound was obtained as a mixture of isomers following theprocedure described for the preparation of intermediate ethyl3-[2-({4-[(tert-butoxycarbonyl)amino]cyclohexyl}amino)-4-cyanophenyl]propanoate(see compound 206, step 3) using ethyl(2E)-3-[3-({4-[(tert-butoxycarbonyl)amino]cyclohexyl}amino)-5-fluoropyridin-2-yl]prop-2-enoate(97% yield). LC-MS (M−H⁺)=410.5

Step 4—Synthesis of tert-butyl[trans-4-(7-fluoro-2-oxo-3,4-dihydro-1,5-naphthyridin-1(2H)-yl)cyclohexyl]carbamate

3-[3-({4-[(tert-butoxycarbonyl)amino]cyclohexyl}amino)-5-fluoropyridin-2-yl]propanoate(1.8 g, 4.5 mmol) was dissolved in THF (20 mL), t-BuONa (0.87 g, 9 mmol)was added and the mixture was stirred at 50° C. for 4 h. Ethyl acetatewas added followed by water. The organic phase was separated, dried overNa₂SO₄ and evaporated in vacuo. The crude was purified by Si-column (cyto cy/ethyl acetate 1:1) to obtain 180 mg of the title transdiastereoisomer (0.18 g, 0.5 mmol, 11% yield). LC-MS (M−H⁺)=364.4

Step 5—Synthesis of tert-butyl[trans-4-(7-fluoro-2-oxo-1,5-naphthyridin-1(2H)-yl)cyclohexyl]carbamate

The title compound was prepared following the procedure described forthe synthesis of intermediate tert-butyl[trans-4-(7-cyano-2-oxoquinolin-1(2H)-yl)cyclohexyl]carbamate (seecompound 206, step 6) using tert-butyl[trans-4-(7-fluoro-2-oxo-3,4-dihydro-1,5-naphthyridin-1(2H)-yl)cyclohexyl]carbamate(96% yield). LC-MS (M−H⁺)=362.2

Step 6—Synthesis of1-(trans-4-aminocyclohexyl)-7-fluoro-1,5-naphthyridin-2(1H)-one

The title compound was obtained following the procedure described forthe preparation of intermediate1-(trans-4-aminocyclohexyl)-7-fluoro-1H-pyrido[2,3-b][1,4]oxazin-2(3H)-one(see compound 193, step 8) using tert-butyl[trans-4-(7-fluoro-2-oxo-1,5-naphthyridin-1(2H)-yl)cyclohexyl]carbamate(98% yield). LC-MS (M−H⁺)=262.2

Step 7—Synthesis of8-fluoro-2-({[trans-4-(7-fluoro-2-oxo-1,5-naphthyridin-1(2H)-yl)cyclohexyl]amino}methyl)[1]benzopyrano[4,3-b]pyrrol-4(1H)-one(formate salt, compound 207)

The title compound was prepared according to the procedure described forthe synthesis of8-fluoro-2-({[trans-4-(7-fluoro-2-oxo-2,3-dihydro-1H-pyrido[2,3-b][1,4]oxazin-1-yl)cyclohexyl]amino}methyl)[1]benzopyrano[4,3-b]pyrrol-4(1H)-one(compound 193, step 9) using1-(trans-4-aminocyclohexyl)-7-fluoro-1,5-naphthyridin-2(1H)-one (18%yield). LC-MS (M−H⁺)=477.2; ¹H NMR (400 MHz, DMSO-d₆) δ ppm 1.28-1.46(m, 2H), 1.56-1.70 (m, 2H), 1.93-2.12 (m, 2H), 2.57 (br. s., 2H), 3.90(s, 2H), 4.45 (br. s., 1H), 6.59 (s, 1H), 6.72 (d, J=9.29 Hz, 1H), 7.28(td, J=8.80, 2.93 Hz, 1H), 7.46 (dd, J=9.05, 4.65 Hz, 1H), 7.86 (d,J=9.29 Hz, 1H), 7.94 (dd, J=8.80, 2.93 Hz, 1H), 8.18 (s, 1H), 8.28 (d,J=9.78 Hz, 1H), 8.52 (d, J=1.96 Hz, 1H), 12.62 (br. s., 1H).

Preparation of Compound 208

Compound 208 was prepared as described herein below.

Step 1—Synthesis of tert-butyl[trans-4-(5-methoxy-2-nitroanilino)cyclohexyl]carbamate

A suspension of 2-chloro-4-methoxy-1-nitrobenzene (2.0 g, 10.7 mmol),N-Boc-trans-1,4-cyclohexanediamine (2.3 g, 10.7 mmol) and triethylamine(1.8 mL, 12.8 mmol) in acetonitrile (80 mL) was heated at 130° C. in asealed tube for 3 days. The mixture was cooled to room temperature, thevolatiles were removed under vacuum and the crude was purified by columnchromatography (from cyclohexane/EtOAc 85:15 to cyclohexane/EtOAc/MeOH6:3:1) to afford the title compound (1.2 g, 3.3 mmol, 31% yield). LC-MS(M−H⁺)=366.4

Step 2—Synthesis of tert-butyl[trans-4-(2-amino-5-methoxyanilino)cyclohexyl]carbamate

The title compound was prepared according to the procedure described forthe synthesis of tert-butyl4-[(3-amino-6-methoxypyridin-2-yl)amino]piperidine-1-carboxylate (seecompound 197, step 2) using tert-butyl[trans-4-(5-methoxy-2-nitroanilino)cyclohexyl]carbamate and MeOH assolvent (94% yield). LC-MS (M−H⁺)=336.4

Step 3—Synthesis of ethylN-[2-({trans-4-[(tert-butoxycarbonyl)amino]cyclohexyl}amino)-4-methoxyphenyl]glycinate

The title compound was prepared according to the procedure described forthe synthesis of tert-butyl4-({3-[(2-ethoxy-2-oxoethyl)amino]-6-methoxypyridin-2-yl}amino)piperidine-1-carboxylate(see compound 197, step 3) using tert-butyl[trans-4-(2-amino-5-methoxyanilino)cyclohexyl]carbamate (63% yield).LC-MS (M−H⁺)=422.5

Step 4—Synthesis of tert-butyl[trans-4-(7-methoxy-2-oxoquinoxalin-1(2H)-yl)cyclohexyl]carbamate

The title compound was prepared according to the procedure described forthe synthesis of tert-butyl4-(6-methoxy-3-oxo-2,3-dihydropyrido[2,3-b]pyrazin-4(1H)-yl)piperidine-1-carboxylate(see compound 197, step 4) using ethylN-[2-({trans-4-[(tert-butoxycarbonyl)amino]cyclohexyl}amino)-4-methoxyphenyl]glycinate(59% yield). LC-MS (M−H⁺)=374.4

Step 5—Synthesis of1-(trans-4-aminocyclohexyl)-7-methoxyquinoxalin-2(1H)-one

The title compound was prepared according to the procedure described forthe synthesis of1-(trans-4-aminocyclohexyl)-7-fluoro-1H-pyrido[2,3-b][1,4]oxazin-2(3H)-one(see compound 193, step 8) using tert-butyl[trans-4-(7-methoxy-2-oxoquinoxalin-1(2H)-yl)cyclohexyl]carbamate (96%yield). LC-MS (M−H⁺)=274.3

Step 6—Synthesis of8-fluoro-2-({[trans-4-(7-methoxy-2-oxoquinoxalin-1(2H)-yl)cyclohexyl]amino}methyl)[1]benzopyrano[4,3-b]pyrrol-4(1H)-one(formate salt, compound 208)

The title compound was prepared according to the procedure described forthe synthesis of8-fluoro-2-({[trans-4-(7-fluoro-2-oxo-2,3-dihydro-1H-pyrido[2,3-b][1,4]oxazin-1-yl)cyclohexyl]amino}methyl)[1]benzopyrano[4,3-b]pyrrol-4(1H)-one(compound 193, step 9) using1-(trans-4-aminocyclohexyl)-7-methoxyquinoxalin-2(1H)-one (20% yield).LC-MS (M−H⁺)=489.2. ¹H NMR (400 MHz, DMSO-d₆) δ ppm 1.31-1.50 (m, 2H),1.69 (d, J=10.63 Hz, 2H), 2.07 (d, J=10.85 Hz, 2H), 2.52-2.70 (m, 3H),3.91 (s, 3H), 3.94 (s, 2H), 4.58 (br. s., 1H), 6.62 (s, 1H), 7.01 (dd,J=8.82, 2.36 Hz, 1H), 7.20 (br. s., 1H), 7.30 (td, J=8.74, 3.01 Hz, 1H),7.47 (dd, J=9.10, 4.60 Hz, 1H), 7.74 (d, J=8.88 Hz, 1H), 7.88-8.00 (m,2H), 8.18 (s, 1H), 12.72 (br. s., 1H).

Preparation of Compounds 209 and 210

Compounds 209 and 210 were prepared as described herein below.

Step 1—Synthesis of ethyl N-(5-fluoro-3-nitropyridin-2-yl)glycinate

The synthesis was performed according to the procedure described for thepreparation of intermediate tert-butyl4-({3-[(2-ethoxy-2-oxoethyl)amino]-6-methoxypyridin-2-yl}amino)piperidine-1-carboxylate(see compound 197, step 3) usingN-(5-fluoro-3-nitropyridin-2-yl)glycinate (95% yield). LC-MS(M−H⁺)=244.2

Step 2—Synthesis of tert-butyl4-[(3-amino-6-methoxypyridin-2-yl)amino]piperidine-1-carboxylate

The synthesis was performed according to the procedure described for thepreparation of intermediate tert-butyl4-[(3-amino-6-methoxypyridin-2-yl)amino]piperidine-1-carboxylate (seecompound 197, step 2) using 5-fluoro-3-nitropyridin-2-amine (47% yield).LC-MS (M−H⁺)=214.0

Step 3—Synthesis of ethylN-[3-({4-[(tert-butoxycarbonyl)amino]cyclohexyl}amino)-5-fluoropyridin-2-yl]glycinate

The title compound was obtained as a mixture of isomers following theprocedure described for the preparation of intermediate tert-butyl{4-[(5-fluoro-2-hydroxypyridin-3-yl)amino]cyclohexyl}carbamate (seecompound 193, step 6) using tert-butyl4-[(3-amino-6-methoxypyridin-2-yl)amino]piperidine-1-carboxylate (69%yield). LC-MS (M−H⁺)=411.4

Step 4—Synthesis of tert-butyl[4-(7-fluoro-2-oxopyrido[2,3-b]pyrazin-1(2H)-yl)cyclohexyl]carbamate

The title compound was obtained as a mixture of isomers following theprocedure described for the preparation of intermediate tert-butyl4-(6-methoxy-3-oxo-2,3-dihydropyrido[2,3-b]pyrazin-4(1H)-yl)piperidine-1-carboxylate(see compound 197, step 4) using ethylN-[3-({4-[(tert-butoxycarbonyl)amino]cyclohexyl}amino)-5-fluoropyridin-2-yl]glycinate(43% yield). LC-MS (M−H⁺)=363.4

Step 5—Synthesis of1-(4-aminocyclohexyl)-7-fluoropyrido[2,3-b]pyrazin-2(1H)-one

The title compound was obtained as a mixture of isomers following theprocedure described for the preparation of intermediate1-(trans-4-aminocyclohexyl)-7-fluoro-1H-pyrido[2,3-b][1,4]oxazin-2(3H)-one(see compound 193, step 8) using tert-butyl[4-(7-fluoro-2-oxopyrido[2,3-b]pyrazin-1(2H)-yl)cyclohexyl]carbamate(94% yield). LC-MS (M−H⁺)=263.1

Step 6—Synthesis of8-fluoro-2-({[trans-4-(7-fluoro-2-oxopyrido[2,3-b]pyrazin-1(2H)-yl)cyclohexyl]amino}methyl)[1]benzopyrano[4,3-b]pyrrol-4(1H)-one(formate salt, compound 209) and8-fluoro-2-({[cis-4-(7-fluoro-2-oxopyrido[2,3-b]pyrazin-1(2H)-yl)cyclohexyl]amino}methyl)[1]benzopyrano[4,3-b]pyrrol-4(1H)-one(formate salt, compound 210)

The title compounds were prepared according to the procedure describedfor the synthesis of8-fluoro-2-({[trans-4-(7-fluoro-2-oxo-2,3-dihydro-1H-pyrido[2,3-b][1,4]oxazin-1-yl)cyclohexyl]amino}methyl)[1]benzopyrano[4,3-b]pyrrol-4(1H)-one(compound 193, step 9) using1-(4-aminocyclohexyl)-7-fluoropyrido[2,3-b]pyrazin-2(1H)-one as amixture of isomers.

A purification by preparative HPLC (A=0.1% ammonia aqueous solution,B=acetonitrile, form 98:2 A:B to 100% B) afforded the trans (10% yield)and cis (6% yield) diastereoisomers.

Compound 209: LC-MS (M−H⁺)=478.4. ¹H NMR (400 MHz, DMSO-d₆) δ ppm 1.41(m, J=11.80 Hz, 2H), 1.70 (d, J=11.29 Hz, 2H), 2.06 (d, J=10.29 Hz, 2H),2.51-2.58 (m, 2H), 2.67 (br. s., 1H), 3.96 (s, 2H), 4.47-4.79 (m, 1H),6.63 (s, 1H), 7.30 (td, J=8.78, 3.01 Hz, 1H), 7.48 (dd, J=9.03, 4.52 Hz,1H), 7.95 (dd, J=9.03, 3.01 Hz, 1H), 8.21 (s, 1H), 8.29 (s, 1H), 8.45(d, J=11.04 Hz, 1H), 8.58 (d, J=2.26 Hz, 1H).

Compound 210: LC-MS (M−H⁺)=478.4. ¹H NMR (400 MHz, DMSO-d₆) δ ppm 1.50(d, J=9.39 Hz, 2H), 1.67 (t, J=12.91 Hz, 2H), 1.95 (d, J=14.09 Hz, 2H),2.69-2.80 (m, 2H), 2.92 (br. s., 1H), 3.90 (s, 2H), 4.92 (br. s., 1H),6.65 (s, 1H), 7.21-7.31 (m, 1H), 7.45 (dd, J=9.00, 4.70 Hz, 1H), 7.87(dd, J=9.00, 3.13 Hz, 1H), 8.19 (s, 1H), 8.33 (s, 1H), 8.47 (d, J=10.56Hz, 1H), 8.54 (d, J=2.35 Hz, 1H).

Preparation of Compound 211

Compound8-fluoro-2-({[trans-4-(7-fluoro-2-oxoquinoxalin-1(2H)-yl)cyclohexyl]amino}methyl)[1]benzopyrano[4,3-b]pyrrol-4(1H)-one(formate salt, compound 211) was prepared according to the proceduredescribed for the synthesis of compound 208, starting from2,4-difluoro-1-nitrobenzene.

LC-MS (M−H⁺)=477.4. ¹H NMR (400 MHz, DMSO-d₆) δ ppm 1.32-1.50 (m, 2H),1.68 (d, J=10.56 Hz, 2H), 2.05 (d, J=11.35 Hz, 2H), 2.37-2.64 (m, 3H),3.93 (s, 2H), 4.56 (br. s., 1H), 6.62 (s, 1H), 7.24 (td, J=8.41, 2.35Hz, 1H), 7.29 (td, J=8.71, 2.93 Hz, 1H), 7.47 (dd, J=9.00, 4.70 Hz, 1H),7.86 (dd, J=8.80, 6.46 Hz, 2H), 7.95 (dd, J=9.19, 2.93 Hz, 1H), 8.06 (s,1H), 8.23 (s, 1H), 10.48-13.49 (m, 2H).

Preparation of Compounds 212 and 213

Compounds 212 and 213 were prepared as described herein below.

Step 1—Synthesis of tert-butyl[4-(7-methoxy-2-oxopyrido[2,3-b]pyrazin-1(2H)-yl)cyclohexyl]carbamate

200 mg (0.55 mmol) of tert-butyl[4-(7-fluoro-2-oxopyrido[2,3-b]pyrazin-1(2H)-yl)cyclohexyl]carbamate,prepared as previously described (step 4 in the synthesis of compounds209 and 210, mixture of isomers), was dissolved in MeOH (10 mL). NaOMe(25% wt solution in MeOH, 25 mL) was added and the mixture was stirredat 50° C. for 3 h. The solvent was evaporated in vacuo, DCM was addedand the solution was washed with sat. NaHCO₃. The organic phase wasseparated, dried over Na₂SO₄ and concentrated in vacuo to obtain 210 mg(0.55 mmol, quant. yield) of the title product, that was progressedwithout any further purification. LC-MS (M−H⁺)=375.4

Step 2—Synthesis of1-(4-aminocyclohexyl)-7-methoxypyrido[2,3-b]pyrazin-2(1H)-one

The title compound was obtained as a mixture of isomers following theprocedure described for the preparation of intermediate1-(trans-4-aminocyclohexyl)-7-fluoro-1H-pyrido[2,3-b][1,4]oxazin-2(3H)-one(see compound 193, step 8) using tert-butyl[4-(7-methoxy-2-oxopyrido[2,3-b]pyrazin-1(2H)-yl)cyclohexyl]carbamate(82% yield). LC-MS (M−H⁺)=275.2

Step 3—Synthesis of8-fluoro-2-({[trans-4-(7-methoxy-2-oxopyrido[2,3-b]pyrazin-1(2H)-yl)cyclohexyl]amino}methyl)[1]benzopyrano[4,3-b]pyrrol-4(1H)-one(hydrochloride, compound 212) and8-fluoro-2-({[cis-4-(7-methoxy-2-oxopyrido[2,3-b]pyrazin-1(2H)-yl)cyclohexyl]amino}methyl)[1]benzopyrano[4,3-b]pyrrol-4(1H)-one(hydrochloride, compound 213)

The title compounds were prepared according to the procedure describedfor the synthesis of8-fluoro-2-({[trans-4-(7-fluoro-2-oxo-2,3-dihydro-1H-pyrido[2,3-b][1,4]oxazin-1-yl)cyclohexyl]amino}methyl)[1]benzopyrano[4,3-b]pyrrol-4(1H)-one(compound 193, step 9) using1-(4-aminocyclohexyl)-7-methoxypyrido[2,3-b]pyrazin-2(1H)-one as amixture of the isomers.

A purification by preparative HPLC (A=0.1% ammonia aqueous solution,B=acetonitrile, form 98:2 A:B to 100% B) followed by the formation ofthe hydrochloride salt under standard conditions afforded the trans (43%yield) and cis (10% yield) diastereoisomers.

Compound 212: LC-MS (M−H⁺)=490.1. ¹H NMR (400 MHz, DMSO-d₆) δ ppm1.69-1.79 (m, 2H), 1.84 (d, J=10.98 Hz, 2H), 2.25-2.34 (m, 2H),2.56-2.71 (m, 2H), 3.17-3.35 (m, 1H), 4.03 (s, 3H), 4.37-4.45 (m, 2H),4.67 (br. s., 1H), 6.96 (d, J=1.65 Hz, 1H), 7.38 (td, J=8.78, 3.02 Hz,1H), 7.53 (dd, J=9.19, 4.53 Hz, 1H), 7.69-7.81 (m, 1H), 7.88 (dd,J=8.65, 2.88 Hz, 1H), 8.16 (s, 1H), 8.35 (d, J=2.47 Hz, 1H), 9.42 (br.s., 2H), 13.54 (br. s., 1H).

Compound 213: LC-MS (M−H⁺)=490.1. ¹H NMR (400 MHz, DMSO-d₆) δ ppm 1.69(d, J=10.96 Hz, 2H), 2.01 (m, J=13.59 Hz, 2H), 2.18 (d, J=14.03 Hz, 2H),2.66-2.80 (m, 2H), 3.49 (br. s., 1H), 4.03 (s, 3H), 4.40-4.45 (m, 2H),4.62-4.78 (m, 1H), 6.99 (d, J=1.75 Hz, 1H), 7.38 (td, J=8.77, 3.07 Hz,1H), 7.53 (dd, J=9.21, 4.38 Hz, 1H), 7.76 (d, J=2.19 Hz, 1H), 7.87 (dd,J=8.33, 3.07 Hz, 1H), 8.18 (s, 1H), 8.36 (d, J=2.19 Hz, 1H), 9.25 (br.s., 2H), 13.49 (s, 1H).

Preparation of Compounds 214 and 215

Compounds 214 and 215 were prepared as described herein below.

Step 1—Synthesis of (aminooxy)(6-hydroxy-5-nitropyridin-3-yl)methanone

6-hydroxy-5-nitropyridine-3-carboxylic acid (3.3 g, 17.9 mmol) wasdissolved in DMF (16 mL). CDI (3.2 g, 19.7 mmol) was added portionwiseat room temperature. The resulting mixture was stirred for 2 hours at60° C. then was cooled to room temperature. 30% aqueous ammonia (23 mL)was added and the resulting mixture was stirred for 20 min. Theprecipitate was filtered, washed with water (5 mL) and dried to give thetitle product (2.85 g, 15.5 mmol, 86% yield), that was progressedwithout any further purification. LC-MS (M−H⁺)=184.1

Step 2—Synthesis of 6-hydroxy-5-nitropyridine-3-carbonitrile

Phosphorus(V) oxychloride (0.66 mL, 7.1 mmol) was added to DMF (11 mL)at 0° C. and the resulting mixture was stirred at the same temperaturefor 15 min. Intermediate(aminooxy)(6-hydroxy-5-nitropyridin-3-yl)methanone (1.1 g, 6.0 mmol) wasadded portionwise. The reaction mixture was heated at 50° C. for 1 hourthen was cooled to 0° C. Water (12 mL) was added, the precipitate wascollected by filtration and washed with additional water (6 mL).Acetonitrile was added to the solid and evaporated under vacuum severaltimes to remove residual water. The obtained title compound (0.74 g, 4.5mmol, 75% yield) was progressed without any further purification. LC-MS(M−H⁺)=166.1

Step 3—Synthesis of 5-amino-6-hydroxypyridine-3-carbonitrile

The title compound was prepared according to the procedure described forthe synthesis of tert-butyl4-[(3-amino-6-methoxypyridin-2-yl)amino]piperidine-1-carboxylate (seecompound 197, step 2) using 6-hydroxy-5-nitropyridine-3-carbonitrile andMeOH/DMF as solvents (96% yield). LC-MS (M−H⁺)=136.1

Step 4—Synthesis of tert-butyl{4-[(5-cyano-2-hydroxypyridin-3-yl)amino]cyclohexyl}carbamate

The title compound was obtained as a mixture of isomers following theprocedure described for the preparation of intermediate tert-butyl{4-[(5-fluoro-2-hydroxypyridin-3-yl)amino]cyclohexyl}carbamate (seecompound 193, step 6) using 5-amino-6-hydroxypyridine-3-carbonitrile(15% yield). LC-MS (M−H⁺)=333.3

Step 5—Synthesis of tert-butyl[4-(7-cyano-2-oxo-2,3-dihydro-1H-pyrido[2,3-b][1,4]oxazin-1-yl)cyclohexyl]carbamate

DIPEA (4.0 mL, 23.4 mmol) and chloroacetyl chloride (934 μL, 11.7 mmol)were added to a stirred solution of tert-butyl{4-[(5-cyano-2-hydroxypyridin-3-yl)amino]cyclohexyl}carbamate (390 mg,1.17 mmol) in DMF (24 mL). The reaction mixture was heated at 150° C. ina sealed flask for 1 hour then was cooled and partitioned between EtOAcand a 1/1 mixture of brine and sat. NaHCO₃. The aqueous layer wasextracted three times with EtOAc and the combined organic layers werewashed with brine. The volatiles were removed under vacuum and theresidue was purified by column chromatography on silica gel (fromcyclohexane/EtOAc 85:15 to cyclohexane/EtOAc/MeOH 6:3:1). A furtherpurification through column chromatography on NH—SiO₂ (cyclohexane/EtOAcfrom 6:4 to 0:100) afforded the title product as a mixture of isomers(208 mg, 1 mmol, 48% yield). LC-MS (M−H⁺)=373.3

Step 6—Synthesis of1-(4-aminocyclohexyl)-2-oxo-2,3-dihydro-1H-pyrido[2,3-b][1,4]oxazine-7-carbonitrile

The title intermediate was prepared as a mixture of isomers according tothe procedure described for the synthesis of1-(trans-4-aminocyclohexyl)-7-fluoro-1H-pyrido[2,3-b][1,4]oxazin-2(3H)-one(see compound 193, step 8) using tert-butyl[4-(7-cyano-2-oxo-2,3-dihydro-1H-pyrido[2,3-b][1,4]oxazin-1-yl)cyclohexyl]carbamate(98% yield). LC-MS (M−H⁺)=273.3

Step 7—Synthesis of1-(trans-4-{[(8-fluoro-4-oxo-1,4-dihydro[1]benzopyrano[4,3-b]pyrrol-2-yl)methyl]amino}cyclohexyl)-2-oxo-2,3-dihydro-1H-pyrido[2,3-b][1,4]oxazine-7-carbonitrile(formate salt, compound 214) and1-(cis-4-{[(8-fluoro-4-oxo-1,4-dihydro[1]benzopyrano[4,3-b]pyrrol-2-yl)methyl]amino}cyclohexyl)-2-oxo-2,3-dihydro-1H-pyrido[2,3-b][1,4]oxazine-7-carbonitrile(formate salt, compound 215)

The title compounds were prepared according to the procedure describedfor the synthesis of8-fluoro-2-({[trans-4-(7-fluoro-2-oxo-2,3-dihydro-1H-pyrido[2,3-b][1,4]oxazin-1-yl)cyclohexyl]amino}methyl)[1]benzopyrano[4,3-b]pyrrol-4(1H)-one(compound 193, step 9) using1-(4-aminocyclohexyl)-2-oxo-2,3-dihydro-1H-pyrido[2,3-b][1,4]oxazine-7-carbonitrileas mixture of isomers.

A purification by preparative HPLC (A=0.1% ammonia aqueous solution,B=acetonitrile, form 98:2 A:B to 100% B) followed by the formation ofthe formate salts under standard conditions afforded the trans (21%yield) and cis (15% yield) diastereoisomers.

Compound 214: LC-MS (M−H⁺)=488.2. ¹H NMR (400 MHz, DMSO-d₆) δ ppm 1.35(q, J=11.74 Hz, 2H), 1.75 (d, J=10.96 Hz, 2H), 2.06 (d, J=11.35 Hz, 2H),2.25-2.40 (m, 2H), 2.61-2.71 (m, 1H), 3.99 (s, 2H), 4.04-4.14 (m, 1H),4.83 (s, 2H), 6.65 (s, 1H), 7.30 (td, J=8.71, 2.93 Hz, 1H), 7.47 (dd,J=9.00, 4.70 Hz, 1H), 7.95 (dd, J=9.00, 3.13 Hz, 1H), 8.21 (s, 1H), 8.31(d, J=1.56 Hz, 1H), 8.35 (d, J=1.96 Hz, 1H), 12.35-13.58 (m, 1H).

Compound 215: LC-MS (M−H⁺)=488.2. ¹H NMR (400 MHz, DMSO-d₆) δ ppm 1.46(d, J=10.27 Hz, 2H), 1.57 (t, J=13.69 Hz, 2H), 1.86 (d, J=13.69 Hz, 2H),2.51-2.61 (m, 2H), 2.81 (br. s., 1H), 3.86 (s, 2H), 4.29 (t, J=11.98 Hz,1H), 4.87 (s, 2H), 6.61 (s, 1H), 7.28 (td, J=8.80, 2.93 Hz, 1H), 7.46(dd, J=9.05, 4.65 Hz, 1H), 7.87 (dd, J=9.05, 3.18 Hz, 1H), 8.17 (s, 1H),8.30 (d, J=1.96 Hz, 1H), 8.35 (d, J=1.96 Hz, 1H), 12.58 (br. s., 1H).

Preparation of Compound 216

Compound8-fluoro-2-({[trans-4-(7-fluoro-2-oxoquinolin-1(2H)-yl)cyclohexyl]amino}methyl)[1]benzopyrano[4,3-b]pyrrol-4(1H)-one(hydrochloride, compound 216) was prepared according to the proceduredescribed for the synthesis of compound 206, starting from5-fluoro-2-iodoaniline.

LC-MS (M−H⁺)=476.1. ¹H NMR (400 MHz, DMSO-d₆) δ ppm 1.43-1.79 (m, 4H),2.18 (br. s., 2H), 2.57-2.77 (m, 2H), 2.80-3.13 (m, 1H), 4.19 (br. s.,2H), 4.32-5.63 (m, 1H), 6.47 (d, J=5.87 Hz, 1H), 6.81 (br. s., 1H), 7.13(td, J=8.44, 2.20 Hz, 1H), 7.34 (td, J=8.56, 2.93 Hz, 1H), 7.51 (dd,J=9.05, 4.65 Hz, 1H), 7.66-7.81 (m, 2H), 7.86 (d, J=9.29 Hz, 1H), 7.91(dd, J=8.80, 2.93 Hz, 1H), 8.32-10.31 (m, 1H), 13.13 (br. s., 1H).

Preparation of Compound 217

Compound 217 was prepared as described herein below.

Step 1—Synthesis of diethyl1-[(diphenylmethylidene)amino]-4-oxocyclohexane-1,3-dicarboxylate

Ethyl N-(diphenylmethylidene)glycinate (10 g, 37 mmol) was dissolved inTHF (100 mL) and the solution was cooled at 0° C. under a N₂ atmosphere.t-BuOK (20.9 g, 187 mmol) was added and the mixture was stirred at 0° C.for 20 min. Then ethyl acrylate (20.3 mL, 187 mmol) was added dropwiseand the reaction mixture was stirred at 0° C. for 3 h. A saturatedsolution of NH₄Cl was added, the organic phase was separated, dried overNa₂SO₄ and concentrated in vacuo. The residue containing the titleintermediate was progressed into the next step without furtherpurification and characterization.

Step 2—Synthesis of diethyl1-[(tert-butoxycarbonyl)amino]-4-oxocyclohexane-1,3-dicarboxylate

The residue from step 1 was dissolved in THF (80 mL) and treated withHCl (2 M solution, 50 mL). Ethyl acetate was added, the organic layerwas discarded and the aqueous phase was basified with K₂CO₃ (pH 8). THFwas added followed by di-tert-butyl dicarbonate (1 eq.) and theresulting mixture was stirred at room temperature overnight. Ethylacetate was added, the organic phase was separated, dried over Na₂SO₄and evaporated in vacuo. The crude material was purified by Si-column(cy to cy/EtOAc 1:1) to obtain 2.8 g (7.8 mmol, 21% yield over twosteps) of the title product. LC-MS (M−H⁺)=358.4

Step 3—Synthesis of ethyl1-[(tert-butoxycarbonyl)amino]-4-oxocyclohexane-1-carboxylate

Diethyl1-[(tert-butoxycarbonyl)amino]-4-oxocyclohexane-1,3-dicarboxylate (300mg, 0.84 mmol) was dissolved in DMSO (1 mL), sodium chloride (200 mg,3.4 mmol) and water (0.05 mL) were added and the reaction mixture wasstirred at 150° C. for 4 h. The mixture was cooled and diluted withethyl acetate. The organic layer was washed with water, dried over MgSO₄and concentrated under reduced pressure. The residue was purified bySi-column (cy to cy/EtOAc 7:3) to obtain 185 mg (0.64 mmol, 76% yield)of the title product. LC-MS (M−H⁺)=286.3

Step 4—Synthesis of ethyltrans-1-[(tert-butoxycarbonyl)amino]-4-[(5-fluoro-2-hydroxypyridin-3-yl)amino]cyclohexane-1-carboxylate

The synthesis was performed according to the procedure described for thepreparation of intermediate tert-butyl{4-[(5-fluoro-2-hydroxypyridin-3-yl)amino]cyclohexyl}carbamate (seecompound 193, step 6) using ethyl1-[(tert-butoxycarbonyl)amino]-4-oxocyclohexane-1-carboxylate. Apurification by C-18 chromatography (from water+0.1% formic acid towater+0.1% formic acid/acetonitrile+0.1% formic acid 8:2) provided thetitle compound (25% yield). LC-MS (M−H⁺)=398.4

Step 5—Synthesis oftrans-1-[(tert-butoxycarbonyl)amino]-4-[(5-fluoro-2-hydroxypyridin-3-yl)amino]cyclohexane-1-carboxylicacid

The synthesis was performed according to the procedure described for thepreparation of intermediate3-[2-({4-[(tert-butoxycarbonyl)amino]cyclohexyl}amino)-4-cyanophenyl]propanoicacid (see compound 206, step 4) using ethyltrans-1-[(tert-butoxycarbonyl)amino]-4-[(5-fluoro-2-hydroxypyridin-3-yl)amino]cyclohexane-1-carboxylate(98% yield). LC-MS (M−H)=368.4

Step 6—Synthesis of tert-butyl{trans-1-carbamoyl-4-[(5-fluoro-2-hydroxypyridin-3-yl)amino]cyclohexyl}carbamate

Intermediatetrans-1-[(tert-butoxycarbonyl)amino]-4-[(5-fluoro-2-hydroxypyridin-3-yl)amino]cyclohexane-1-carboxylicacid (340 mg, 0.9 mmol) was dissolved in DMF (20 mL). DIPEA (0.8 mL, 4.6mmol) and ammonium chloride (199 mg, 3.7 mmol) were added and themixture was cooled to 0° C. HATU (426 mg, 1.1 mmol) was added at thesame temperature and the mixture was allowed to slowly reach roomtemperature. After 6 hours water was added, the mixture was extractedwith ethyl acetate, the organic phase was separated, dried over Na₂SO₄and concentrated in vacuo. The resulting crude material was purified bySi-column (from DCM to DCM/MeOH 8:2) to obtain the title compound (233mg, 0.63 mmol, 68% yield). LC-MS (M−H⁻)=369.3

Step 7—Synthesis of tert-butyl[trans-1-carbamoyl-4-(7-fluoro-2-oxo-2,3-dihydro-1H-pyrido[2,3-b][1,4]oxazin-1-yl)cyclohexyl]carbamate

The synthesis was performed according to the procedure described for thepreparation of intermediate tert-butyl[4-(7-fluoro-2-oxo-2,3-dihydro-1H-pyrido[2,3-b][1,4]oxazin-1-yl)cyclohexyl]carbamate(see compound 193, step 7) using tert-butyl{trans-1-carbamoyl-4-[(5-fluoro-2-hydroxypyridin-3-yl)amino]cyclohexyl}carbamate(47% yield). LC-MS (M−H⁺)=409.2

Step 8—Synthesis oftrans-1-amino-4-(7-fluoro-2-oxo-2,3-dihydro-1H-pyrido[2,3-b][1,4]oxazin-1-yl)cyclohexane-1-carboxamide

The synthesis was performed according to the procedure described for thepreparation of intermediate1-(trans-4-aminocyclohexyl)-7-fluoro-1H-pyrido[2,3-b][1,4]oxazin-2(3H)-one(see compound 193, step 8) using tert-butyl[trans-1-carbamoyl-4-(7-fluoro-2-oxo-2,3-dihydro-1H-pyrido[2,3-b][1,4]oxazin-1-yl)cyclohexyl]carbamate(62% yield). LC-MS (M−H⁺)=309.2

Step 9—Synthesis oftrans-1-{[(8-fluoro-4-oxo-1,4-dihydro[1]benzopyrano[4,3-b]pyrrol-2-yl)methyl]amino}-4-(7-fluoro-2-oxo-2,3-dihydro-1H-pyrido[2,3-b][1,4]oxazin-1-yl)cyclohexane-1-carboxamide(formate salt, compound 217)

The title compound was prepared according to the procedure described forthe synthesis of8-fluoro-2-({[trans-4-(7-fluoro-2-oxo-2,3-dihydro-1H-pyrido[2,3-b][1,4]oxazin-1-yl)cyclohexyl]amino}methyl)[1]benzopyrano[4,3-b]pyrrol-4(1H)-one(compound 193, step 9) usingtrans-1-amino-4-(7-fluoro-2-oxo-2,3-dihydro-1H-pyrido[2,3-b][1,4]oxazin-1-yl)cyclohexane-1-carboxamide(10% yield). LC-MS (M−H⁺)=524.3. ¹H NMR (500 MHz, DMSO-d₆) δ ppm 1.53(td, J=13.21, 3.91 Hz, 2H), 1.57-1.66 (m, 2H), 2.24 (d, J=12.23 Hz, 2H),2.41-2.56 (m, 2H), 3.72 (s, 2H), 4.27-4.54 (m, 1H), 4.74 (s, 2H), 6.58(s, 1H), 7.27-7.33 (m, 2H), 7.48 (dd, J=9.05, 4.65 Hz, 1H), 7.53 (s,1H), 7.82 (dd, J=10.03, 2.69 Hz, 1H), 7.86 (d, J=2.45 Hz, 1H), 7.94 (dd,J=9.05, 3.18 Hz, 1H), 8.18 (s, 1H), 12.40 (br. s., 1H).

Preparation of Compound 219

Compound8-fluoro-2-({[trans-4-(7-methoxy-2-oxoquinolin-1(2H)-yl)cyclohexyl]amino}methyl)[1]benzopyrano[4,3-b]pyrrol-4(1H)-one(formate salt, compound 219) was prepared according to the proceduredescribed for the synthesis of compound 206, starting from2-bromo-5-methoxyaniline.

LC-MS (M−H⁺)=488.3. ¹H NMR (400 MHz, DMSO-d₆) δ ppm 1.32-1.48 (m, 2H),1.64 (d, J=10.74 Hz, 2H), 2.09 (d, J=8.11 Hz, 2H), 2.53-2.76 (m, 3H),3.87 (s, 3H), 3.95 (s, 2H), 4.38 (br. s., 1H), 6.32 (d, J=7.78 Hz, 1H),6.62 (s, 1H), 6.90 (dd, J=8.61, 2.03 Hz, 1H), 7.11 (d, J=1.75 Hz, 1H),7.30 (td, J=8.74, 3.01 Hz, 1H), 7.48 (dd, J=9.10, 4.71 Hz, 1H), 7.62 (d,J=8.66 Hz, 1H), 7.74 (d, J=9.32 Hz, 1H), 7.96 (dd, J=9.10, 2.96 Hz, 1H),8.18 (s, 1H), 12.83 (br. s., 1H).

Preparation of Compound 220

Compound 220 was prepared as described herein below.

Step 1—Synthesis of tert-butyl{trans-4-[(5-fluoro-2-hydroxypyridin-3-yl)amino]-1-(hydroxymethyl)cyclohexyl}carbamate

1.0 g (2.5 mmol) of intermediate ethyltrans-1-[(tert-butoxycarbonyl)amino]-4-[(5-fluoro-2-hydroxypyridin-3-yl)amino]cyclohexane-1-carboxylate,prepared as described in step 4 of the synthesis of compound 217, wasdissolved in THF (20 mL). The solution was cooled to −10° C. then LiAlH₄(1 M in THF, 2.5 mL, 2.5 mmol) was added dropwise. After stirring for 2h the reaction was quenched by adding Na₂SO₄.10H₂O. The mixture wasfiltered and concentrated in vacuo. The crude material was dissolved inethyl acetate and washed with sat. NH₄Cl. The organic phase wasseparated, dried over Na₂SO₄ and concentrated in vacuo to obtain thetitle product (614 mg, 1.8 mmol, 69% yield). LC-MS (M−H⁺)=356.2

Step 2—Synthesis of tert-butyl{trans-1-({[tert-butyl(dimethyl)silyl]oxy}methyl)-4-[(5-fluoro-2-hydroxypyridin-3-yl)amino]cyclohexyl}carbamate

To a solution of tert-butyl{trans-4-[(5-fluoro-2-hydroxypyridin-3-yl)amino]-1-(hydroxymethyl)cyclohexyl}carbamate(344 mg, 0.96 mmol) in DMF (5 mL), imidazole (165 mg, 2.14 mmol) wasadded followed by TBDMSCl (581 mg, 3.87 mmol). The mixture was stirredat room temperature overnight then ethyl acetate was added followed bywater. The organic phase was separated, dried over Na₂SO₄ andconcentrated in vacuo. The crude material was purified by Si-column(from cy to cy/ethyl acetate 1:1) to obtain the title compound (300 mg,0.64 mmol, 67% yield). LC-MS (M−H⁺)=470.4

Step 3—Synthesis of tert-butyl[trans-1-({[tert-butyl(dimethyl)silyl]oxy}methyl)-4-(7-fluoro-2-oxo-2,3-dihydro-1H-pyrido[2,3-b][1,4]oxazin-1-yl)cyclohexyl]carbamate

To a suspension of tert-butyl{trans-1-({[tert-butyl(dimethyl)silyl]oxy}methyl)-4-[(5-fluoro-2-hydroxypyridin-3-yl)amino]cyclohexyl}carbamate(253 mg, 0.54 mmol) and potassium carbonate (224 mg, 1.62 mmol) in DMF(5.2 mL) chloroacetyl chloride (52 μL, 0.65 mmol) was added. The mixturewas stirred at 80° C. for 6 h then was cooled to 0° C. Sat. NaHCO₃ wasadded followed by ethyl acetate. The organic phase was separated, driedover Na₂SO₄ and the solvents were evaporated in vacuo to afford thetitle intermediate (220 mg, 0.43 mmol, 80% yield), that was progressedwithout any further purification. LC-MS (M−H⁺)=510.4

Step 4—Synthesis of1-[trans-4-amino-4-(hydroxymethyl)cyclohexyl]-7-fluoro-1H-pyrido[2,3-b][1,4]oxazin-2(3H)-one

To a stirred solution of tert-butyl[trans-1-({[tert-butyl(dimethyl)silyl]oxy}methyl)-4-(7-fluoro-2-oxo-2,3-dihydro-1H-pyrido[2,3-b][1,4]oxazin-1-yl)cyclohexyl]carbamate(220 mg, 0.43 mmol) in MeOH (1.7 mL) HCl (4 M in dioxane, 2.74 mL) wasadded. The mixture was stirred at room temperature for 4 h then wasconcentrated under reduced pressure. The residue was purified by SCXcolumn to give the title compound (98 mg, 0.33 mmol, 77% yield). LC-MS(M−H⁺)=296.1

Step 5—Synthesis of8-fluoro-2-({[trans-4-(7-fluoro-2-oxo-2,3-dihydro-1H-pyrido[2,3-b][1,4]oxazin-1-yl)-1-(hydroxymethyl)cyclohexyl]amino}methyl)[1]benzopyrano[4,3-b]pyrrol-4(1H)-one(hydrochloride, compound 220)

The title compound was prepared according to the procedure described forthe synthesis of8-fluoro-2-({[trans-4-(7-fluoro-2-oxo-2,3-dihydro-1H-pyrido[2,3-b][1,4]oxazin-1-yl)cyclohexyl]amino}methyl)[1]benzopyrano[4,3-b]pyrrol-4(1H)-one(compound 193, step 9) using1-[trans-4-amino-4-(hydroxymethyl)cyclohexyl]-7-fluoro-1H-pyrido[2,3-b][1,4]oxazin-2(3H)-one(30% yield). LC-MS (M−H⁺)=511.2. ¹H NMR (400 MHz, DMSO-d₆) δ ppm1.66-1.96 (m, 4H), 2.02-2.19 (m, 2H), 2.46 (br. s., 2H), 3.93 (s, 2H),4.00 (br. s., 1H), 4.36 (br. s., 2H), 4.73 (s, 2H), 6.92 (d, J=1.97 Hz,1H), 7.37 (td, J=8.77, 3.07 Hz, 1H), 7.52 (dd, J=9.21, 4.60 Hz, 1H),7.77-8.03 (m, 3H), 9.04 (br. s., 2H), 13.49 (br. s., 1H).

Preparation of Compound 221

Compound 221 was prepared as described herein below.

Step 1—Synthesis of8-fluoro-2-({[trans-4-(7-fluoro-2-oxo-2,3-dihydro-1H-pyrido[2,3-b][1,4]oxazin-1-yl)cyclohexyl]amino}methyl)-1-{[2-(trimethylsilyl)ethoxy]methyl}[1]benzopyrano[4,3-b]pyrrol-4(1H)-one

1-(4-aminocyclohexyl)-7-fluoro-1H,2H,3H-pyrido[2,3-b][1,4]oxazin-2-one(133 mg, 0.5 mmol, see step 8 in the synthesis of compound 193) wassuspended in dry dicloroethane (20 mL).8-fluoro-4-oxo-1-{[2-(trimethylsilyl)ethoxy]methyl}-1H,4H-chromeno[4,3-b]pyrrole-2-carbaldehyde(200 mg, 0.55 mmol, see step 1 in the synthesis of compound 194) wasadded at room temperature followed by glacial acetic acid (catalytic, 2drops). The reaction mixture was stirred at 50° C. for 4 hours then wascooled down to room temperature. Sodium triacetoxyborohydride (264 mg,1.25 mmol) was added and the resulting suspension was stirred overnightat room temperature. The reaction mixture was partitioned betweendichloromethane and a saturated aqueous NaHCO₃ solution. The two layerswere separated and the organic phase was dried over Na₂SO₄, filtered andconcentrated in vacuo. The residue was purified by flash chromatography(Silica N—H, 28 g cartridge, cyclohexane/ethyl acetate from 70:30 to50:50) to afford the title compound (127 mg, 0.2 mmol, 41% yield) as anoff-white solid. LC-MS (M−H⁺)=611.3

Step 2—Synthesis of2-({(2-{[tert-butyl(dimethyl)silyl]oxy}ethyl)[trans-4-(7-fluoro-2-oxo-2,3-dihydro-1H-pyrido[2,3-b][1,4]oxazin-1-yl)cyclohexyl]amino}methyl)-8-fluoro-1-{[2-(trimethylsilyl)ethoxy]methyl}[1]benzopyrano[4,3-b]pyrrol-4(1H)-one

To a solution of8-fluoro-2-({[trans-4-(7-fluoro-2-oxo-2,3-dihydro-1H-pyrido[2,3-b][1,4]oxazin-1-yl)cyclohexyl]amino}methyl)-1-{[2-(trimethylsilyl)ethoxy]methyl}[1]benzopyrano[4,3-b]pyrrol-4(1H)-one(56 mg, 0.09 mmol) in dry dichloroethane (2 mL)2-[(tert-butyldimethylsilyl)oxy]acetaldehyde (0.034 mL, 0.18 mmol) andglacial acetic acid (1 drop) were added. The reaction mixture was heatedat 40° C. for 1 h then NaBH(OAc)₃ (38 mg, 0.18 mmol) was added and theresulting mixture was stirred overnight. The mixture was partitionedbetween dichloromethane and a saturated aq. solution of NaHCO₃. The twolayers were separated and the organic phase was dried over Na₂SO₄,filtered and concentrated in vacuo. The crude material was elutedthrough a 1 g SCX cartridge with methanol and a 2 N solution of NH₃ inmethanol. Evaporation of the solvents afforded the title intermediate(50 mg, 0.065 mmol, 72% yield), which was progressed into the next stepwithout any further purification. LC-MS (M−H⁺)=769.6

Step 3—Synthesis of8-fluoro-2-({[trans-4-(7-fluoro-2-oxo-2,3-dihydro-1H-pyrido[2,3-b][1,4]oxazin-1-yl)cyclohexyl](2-hydroxyethyl)amino}methyl)[1]benzopyrano[4,3-b]pyrrol-4(1H)-one(formate salt, compound 221)

The synthesis was performed according to the procedure described for thepreparation of intermediate1-(trans-4-aminocyclohexyl)-7-fluoro-1H-pyrido[2,3-b][1,4]oxazin-2(3H)-one(see compound 193, step 8) using2-({(2-{[tert-butyl(dimethyl)silyl]oxy}ethyl)[trans-4-(7-fluoro-2-oxo-2,3-dihydro-1H-pyrido[2,3-b][1,4]oxazin-1-yl)cyclohexyl]amino}methyl)-8-fluoro-1-{[2-(trimethylsilyl)ethoxy]methyl}[1]benzopyrano[4,3-b]pyrrol-4(1H)-one(77% yield). LC-MS (M−H⁺)=525.3. ¹H NMR (400 MHz, DMSO-d₆) δ ppm1.58-2.45 (m, 8H), 2.83-2.91 (m, 2H), 3.52 (br. s., 2H), 3.88-4.17 (m,4H), 4.64-4.72 (m, 2H), 6.68 (br. s., 1H), 7.26 (td, J=8.71, 2.93 Hz,1H), 7.44 (dd, J=9.00, 4.70 Hz, 1H), 7.74 (dd, J=9.98, 2.54 Hz, 1H),7.80 (d, J=2.74 Hz, 1H), 7.92 (dd, J=9.00, 3.13 Hz, 1H), 8.12 (s, 1H).

Preparation of Compound 301

Compound 301 was prepared as described herein below.

Step 1—Synthesis of ethyl 3-(3-hydroxypyridin-2-yl)-3-oxopropanoate

1-(3-Hydroxypyridin-2-yl)ethan-1-one (2.50 g, 18.2 mmol) in a mixture ofdiethyl carbonate (20 mL) and toluene (20 mL) was treated portionwisewith NaH (60%, 3.3 g, 82 mmol) and stirred at RT for 1 h. A furtherportion of toluene (10 mL) was added and stirring continued for afurther 5 h. The reaction mixture was added to a mixture of sat. aq.NH₄Cl and EtOAc. The aqueous phase was extracted with EtOAc and thecombined organic fractions were washed with brine, dried over Na₂SO₄ andevaporated. The residue was purified by FCC (0-10% EtOAc in DCM) toafford the title compound (3.27 g, 86% yield) as a pale yellow oil.LC-MS (M−H⁺)=210. ¹H NMR (400 MHz, CDCl₃): δ 11.28 (s, 1H), 8.22 (dd,J=1.4, 4.2 Hz, 1H), 7.43 (dd, J=4.2, 8.5 Hz, 1H), 7.35 (dd, J=1.4, 8.5Hz, 1H), 4.22 (s, 2H), 4.21 (q, J=7.1 Hz, 2H), 1.26 (t, J=7.1 Hz, 3H).

Step 2—Synthesis of 4-hydroxy-2H-pyrano[3,2-b]pyridin-2-one

Ethyl 3-(3-hydroxypyridin-2-yl)-3-oxopropanoate (3.27 g, 15.6 mmol) inxylene (100 mL) was treated with NaH (60%, 63 mg, 1.6 mmol). Thereaction mixture was heated to reflux and some solvent (15 mL) wasdistilled off. Heating under reflux was continued for 7 h. After coolingto RT the solid was collected by filtration, washed with toluene anddried under vacuum to afford the title compound (2.16 g, 85% yield) as abrown solid. LC-MS (M−H⁺)=164. ¹H NMR (400 MHz, DMSO-d6): δ 8.55 (dd,J=1.2, 4.4 Hz, 1H), 7.80 (dd, J=1.2, 8.4 Hz, 1H), 7.64 (dd, J=4.4, 8.4Hz, 1H), 5.58 (s, 1H).

Step 3—Synthesis of4-chloro-2-oxo-2H-pyrano[3,2-b]pyridine-3-carbaldehyde

Phosphorus oxychloride (1.7 mL, 18 mmol) was added to an ice-cooledmixture of 4-hydroxy-2H-pyrano[3,2-b]pyridin-2-one (1.00 g, 6.13 mmol)and DMF (5 mL). The mixture was stirred at RT for 10 min then heated at60° C. for 1.5 h. The reaction was cooled to RT, poured onto ice andextracted with DCM (×3). The combined organic extracts were washed withwater, then dried (Na₂SO₄) and evaporated to afford the title compound(1.69 g, quantitative yield) as a brown solid. LC-MS (M-Cl+H₂O+)=192. ¹HNMR (400 MHz, CDCl₃): δ 10.42 (s, 1H), 8.81 (dd, J=1.4, 4.3 Hz, 1H),7.76 (dd, J=1.4, 8.5 Hz, 1H), 7.69 (dd, J=4.3, 8.5 Hz, 1H).

Step 4—Synthesis of4-oxo-1,4-dihydropyrrolo[2′,3′:4,5]pyrano[3,2-b]pyridine-2-carbaldehyde

4-Chloro-2-oxo-2H-pyrano[3,2-b]pyridine-3-carbaldehyde (76%, 0.513 g,1.86 mmol) was dissolved in ethanol (10 mL) and treated withaminoacetaldehyde dimethylacetal (0.215 g, 2.05 mmol) followed by Et₃N(0.54 mL, 3.9 mmol). The reaction was stirred at 70° C. for 50 min. Thereaction mixture was allowed to cool, toluene was added and thenevaporated. The residue was partitioned between EtOAc and water. Theaqueous phase was extracted with ethyl acetate. The combined organicextracts were washed with brine, dried (Na₂SO₄) and evaporated. Theresulting solid was dissolved in AcOH (20 mL). Water (0.8 mL) was addedand the reaction was stirred at 110° C. for 50 min. The reaction mixturewas allowed to cool, toluene was added and evaporated (×2). The residuewas purified by FCC (EtOAc, then 10% MeOH in DCM) to afford the titlecompound (0.248 g, 62% yield) as a yellow solid. LC-MS (M−H⁺)=215.

Step 5—Synthesis of2-(trans-4-(7-fluoro-2-oxo-2,3-dihydro-1H-pyrido[2,3-b][1,4]oxazin-1-yl)cyclohexyl]amino}methyl)pyrrolo[2′,3′:4,5]pyrano[3,2-b]pyridin-4(1H)-one(compound 301)

The title compound was prepared according to the procedure described forthe synthesis of8-fluoro-2-({[trans-4-(7-fluoro-2-oxo-2,3-dihydro-1H-pyrido[2,3-b][1,4]oxazin-1-yl)cyclohexyl]amino}methyl)[1]benzopyrano[4,3-b]pyrrol-4(1H)-one(compound 193, step 9) using4-oxo-1,4-dihydropyrrolo[2′,3′:4,5]pyrano[3,2-b]pyridine-2-carbaldehyde(75% yield). LC-MS (M−H⁺)=464.3. ¹H NMR (400 MHz, DMSO-d6): δ 8.54 (dd,J=1.3, 4.6 Hz, 1H), 7.88-7.83 (m, 3H), 7.50 (dd, J=4.6, 8.4 Hz, 1H),6.63 (s, 1H), 4.68 (s, 2H), 4.06-3.99 (m, 1H), 3.85 (s, 2H), 2.48-2.41(m, 1H), 2.34-2.24 (m, 2H), 1.99-1.93 (m, 2H), 1.72-1.67 (m, 2H),1.30-1.20 (m, 2H).

Preparation of Compound 302

Compound 302 was prepared as described herein below.

Step 1—Synthesis of 2-chloro-5-methoxypyridin-3-ol

2-Chloro-5-methoxypyridin-3-yl boronic acid (1.0 g, 5.35 mmol) wassuspended in methanol (20 mL) and cooled to 0° C. To the resultingsuspension was added dropwise 50% aq. hydrogen peroxide (2.5 mL) and thereaction mixture was stirred for 5 hours at room temperature. Thereaction was cooled to 0° C. and treated with saturated aqueous sodiumthiosulphate (5 mL), followed by water (50 mL) and extracted withdichloromethane. The combined organic extracts were evaporated todryness. The resulting residue was purified by flash columnchromatography, eluting with 0 to 100% ethyl acetate/iso-hexane, toafford the title compound (506 mg, 60% yield). ¹H NMR (400 MHz, DMSO): δ10.75 (s, 1H), 7.67 (d, J=2.8 Hz, 1H), 6.97 (d, J=2.8 Hz, 1H), 3.84 (s,3H).

Step 2—Synthesis of tert-butyl [trans-4-(2-chloroacetamido)cyclohexyl]carbamate

tert-Butyl (trans-4-aminocyclohexyl)carbamate (10.7 g, 50 mmol) wasdissolved in DCM (200 mL), cooled to 0° C. and treated withtriethylamine (6.1 g, 60.4 mmol). Chloroacetyl chloride (5.65 g, 50mmol) was then added dropwise over 10-15 minutes and the reaction wasleft to stir overnight at room temperature. The reaction was quenchedwith water (100 mL), the phases separated and the combined DCM fractionsdried over MgSO₄, filtered and evaporated. The resulting residue waswashed with iso-hexane, filtered and dried in vacuum to give the titlecompound (11.12 g, 76% yield). ¹H NMR (400 MHz, CDCl₃): δ 6.37 (d, J=7.3Hz, 1H), 4.40-4.39 (m, 1H), 4.02 (s, 2H), 3.79-3.69 (m, 1H), 3.50-3.43(m, 1H), 2.08-1.98 (m, 4H), 1.45 (s, 9H), 1.42-1.20 (m, 4H).

Step 3—Synthesis of tert-butyl[trans-4-(7-methoxy-2-oxo-2,3-dihydro-1H-pyrido[2,3-b][1,4]oxazin-1-yl)cyclohexyl]carbamate

2-Chloro-5-methoxypyridin-3-ol (506 mg, 3.18 mmol) was dissolved in DMF(20 mL) and tert-butyl [trans-4-(2-chloroacetamido)cyclohexyl]carbamate(926 mg, 1 eq) and Cs₂CO₃ (2.5 g, 2.4 eq) added. The reaction mixturewas heated at 120° C. for 18 h. After cooling to room temperature, themixture was filtered and evaporated to dryness. The residue was purifiedby flash column chromatography eluting with 0 to 100% ethylacetate/iso-hexane to afford the title compound (756 mg, 63% yield).LC-MS (M−H⁺)=378. ¹H NMR (400 MHz, DMSO): δ 7.63 (d, J=2.5 Hz, 1H), 7.39(d, J=2.5 Hz, 1H), 6.85 (d, J=7.1 Hz, 1H), 4.68 (s, 2H), 4.16-4.06 (m,1H), 3.90 (s, 3H), 2.47-2.34 (m, 2H), 1.90 (d, J=11.1 Hz, 2H), 1.77 (d,J=10.1 Hz, 2H), 1.44 (s, 11H).

Step 4—Synthesis of1-[trans-4-aminocyclohexyl]-7-methoxy-1H-pyrido[2,3-b][1,4]oxazin-2(3H)-one(hydrochloride salt)

tert-butyl[trans-4-(7-methoxy-2-oxo-2,3-dihydro-1H-pyrido[2,3-b][1,4]oxazin-1-yl)cyclohexyl]carbamate(756 mg, 2.01 mmol) was dissolved in 1,4 dioxane (15 mL). A 4 N solutionof HCl in dioxane (15 mL) was added and the reaction mixture stirred for1 h at room temperature followed by addition of another 15 mL of a 4 Nsolution of HCl in dioxane. The reaction mixture was stirred for 1 h atroom temperature, diluted with diethyl ether and filtered to yield awhite solid. The precipitate was washed with ether and dried in vacuumto give the title compound (675 mg, 96% yield) as HCl salt. ¹H NMR (400MHz, DMSO): δ 8.16 (d, J=1.6 Hz, 3H), 7.60 (d, J=2.5 Hz, 1H), 7.41 (d,J=2.5 Hz, 1H), 4.64 (s, 2H), 4.13-4.05 (m, 1H), 3.85 (s, 3H), 3.11 (d,J=4.4 Hz, 1H), 2.42-2.32 (m, 2H), 2.05 (d, J=11.4 Hz, 2H), 1.78 (d,J=10.5 Hz, 2H), 1.61-1.50 (m, 2H).

Step 5—Synthesis of8-fluoro-2-({[trans-4-(7-methoxy-2-oxo-2,3-dihydro-1H-pyrido[2,3-b][1,4]oxazin-1-yl)cyclohexyl]amino}methyl)[1]benzopyrano[4,3-b]pyrrol-4(1H)-one(compound 302)

1-[trans-4-aminocyclohexyl]-7-methoxy-1H-pyrido[2,3-b][1,4]oxazin-2(3H)-oneHCl (307 mg, 878 μmol) was dissolved in methanol (10 mL) and treatedwith triethylamine (354 mg, 4 eq), followed by 3 Å molecular sieves (500mg) and 8-fluoro-4-oxo-1,4-dihydrochromeno[4,3-b]pyrrole-2-carbaldehyde(231 mg, 1 eq). The reaction mixture was stirred at 60° C. for 18 h,cooled to room temperature and treated with NaBH₄ (134 mg, 4 eq). Theresulting mixture was stirred for 18 h at room temperature then wascooled, filtered through celite and the combined filtrates evaporated.The resulting material was purified by flash column chromatographyeluting with 0 to 100% ethyl acetate/iso-hexane followed by 0 to 100%methanol/ethyl acetate to give 123 mg of crude product. This materialwas triturated with DMF followed by ether, filtered and dried in vacuumto give the title compound (52 mg, 12.1% yield). LC-MS (M−H⁺)=493. ¹HNMR (400 MHz, DMSO): δ 13.35 (bs, 1H), 9.30 (bs, 1H), 7.88 (dd, J=2.6,8.5 Hz, 1H), 7.60 (d, J=2.3 Hz, 1H), 7.53 (dd, J=4.5, 9.1 Hz, 1H),7.44-7.35 (m, 2H), 6.92 (s, 1H), 4.65 (s, 2H), 4.34-4.34 (m, 2H), 4.11(m, 1H), 3.85 (s, 3H), 3.19 (m, 1H), 2.41 (d, J=11.4 Hz, 2H), 2.23 (m,2H), 1.87 (d, J=1.3 Hz, 2H), 1.64-1.63 (m, 2H).

Preparation of Compound 303

Compound 303 was prepared as described herein below.

Step 1—Synthesis of tert-butyl4-(2-chloroacetamido)piperidine-1-carboxylate

To a solution of tert-butyl 4-aminopiperidine-1-carboxylate (2.5 g, 12.5mmol) in dichloromethane (30 mL) and triethylamine (2.25 mL, 16.1 mmol)was added at 0° C. chloroacetyl chloride (1.1 mL, 13.8 mmol). Thereaction mixture was allowed to warm to room temperature and stirringcontinued for 1 hour. The reaction was quenched by addition of asaturated NaHCO₃ solution and extracted with ethyl acetate. The combinedorganic extracts were evaporated and the residue purified by flashcolumn chromatography eluting with ethyl acetate to afford the titlecompound (3.5 g, 100% yield). ¹H NMR (400 MHz, CDCl3): δ 6.46 (d, J=6.1Hz, 1H), 4.08-4.05 (m, 1H), 4.04 (s, 2H), 4.00-3.89 (m, 2H), 2.88 (dd,J=12.2, 12.2 Hz, 2H), 1.96-1.89 (m, 2H), 1.46 (s, 9H), 1.44-1.29 (m,2H).

Step 2—Synthesis of tert-butyl4-(7-fluoro-2-oxo-2,3-dihydro-1H-pyrido[2,3-b][1,4]oxazin-1-yl)piperidine-1-carboxylate

tert-butyl 4-(2-chloroacetamido)piperidine-1-carboxylate (2.02 g, 7.31mmol) and 2-chloro-5-fluoropyridin-3-ol (1.08 g, 7.32 mmol) weredissolved in DMF (70 mL). Caesium carbonate (4.88 g, 15 mmol) was addedand the mixture heated to 100° C. for 18 hours. After cooling to roomtemperature the mixture was partitioned between ethyl acetate and waterand the organic phase washed with water. The organic layer was thenevaporated onto silica and purified by flash column chromatographyeluting with 20 to 100% ethyl acetate/iso-hexane to give the titlecompound (2.19 g, 85% yield). ¹H NMR (400 MHz, CDCl₃): δ 7.80 (d, J=2.5Hz, 1H), 7.23 (dd, J=2.7, 9.0 Hz, 1H), 4.70 (s, 2H), 4.43-4.29 (m, 3H),2.81 (m, 2H), 2.50-2.37 (m, 2H), 1.74 (dd, J=1.5, 11.6 Hz, 2H), 1.50 (s,9H).

Step 3—Synthesis of7-fluoro-1-(piperidin-4-yl)-1H-pyrido[2,3-b][1,4]oxazin-2(3H)-onehydrogen chloride

tert-butyl4-(7-fluoro-2-oxo-2,3-dihydro-1H-pyrido[2,3-b][1,4]oxazin-1-yl)piperidine-1-carboxylate(2.19 g, 6.23 mmol) was dissolved in MeOH (3 mL) and treated with a 2 Nsolution of HCl in diethyl ether (6 mL) at room temperature for 18hours. The mixture was evaporated to furnish the title compound (1.6 g)that was used without further purification in the next step. ¹H NMR (400MHz, DMSO): δ 9.26-9.20 (m, 1H), 8.69-8.67 (m, 1H), 8.02 (dd, J=2.6,10.1 Hz, 1H), 7.90 (d, J=2.5 Hz, 1H), 4.76 (s, 2H), 4.43-4.34 (m, 1H),3.37 (d, J=12.3 Hz, 2H), 3.12-3.00 (m, 2H), 2.79-2.66 (m, 2H), 1.91 (d,J=12.7 Hz, 2H).

Step 4—Synthesis of1-[1-(3-amino-2-hydroxypropyl)piperidin-4-yl]-7-fluoro-1H-pyrido[2,3-b][1,4]oxazin-2(3H)-one

7-Fluoro-1-(piperidin-4-yl)-1H-pyrido[2,3-b][1,4]oxazin-2(3H)-onehydrogen chloride (1.6 g, 6.23 mmol) and2-(oxiran-2-ylmethyl)isoindoline-1,3-dione (1.28 g, 6.3 mmol) weresuspended in acetonitrile (60 mL) and triethylamine (4 mL) was added.The mixture was heated at 70° C. for 20 hours, then cooled to roomtemperature and evaporated to dryness. The residue (2.8 g, 6.23 mmol)was treated with methylamine (20 mL, 33% in ethanol) in a sealed tube at50° C. for 3 hours. The mixture was evaporated and purified by reversephase preparative HPLC to afford the title compound (119 mg). LC-MS(M−H⁺)=325

Step 5—Synthesis of 6-fluoro-2-oxo-2H-1-benzopyran-4-yltrifluoromethanesulfonate

To a solution of 6-fluoro-4-hydroxy-2H-chromen-2-one (720 mg, 4 mmol)and triethylamine (1.4 mL, 10 mmol) in dichloromethane (30 mL),trifluoromethane sulfonic anhydride (0.74 mL, 4.4 mmol) was addeddropwise at 0° C. under nitrogen. The reaction was allowed to warm toroom temperature and stirred overnight. The mixture was then poured intodiethyl ether/iso-hexane (1:1, 600 mL) and filtered through a silicaplug. The filtrate was evaporated to give the title compound (647 mg,52% yield) which was used without further purification in the next step.

Step 6—Synthesis of7-fluoro-1-(1-{3-[(6-fluoro-2-oxo-2H-1-benzopyran-4-yl)amino]-2-hydroxypropyl}piperidin-4-yl)-1H-pyrido[2,3-b][1,4]oxazin-2(3H)-one(compound 303)

1-(1-(3-Amino-2-hydroxypropyl)piperidin-4-yl)-7-fluoro-1H-pyrido[2,3-b][1,4]oxazin-2(3H)-one(95 mg, 277 μmol) and 6-fluoro-2-oxo-2H-chromen-4-yl trifluoromethanesulfonate (320 mg, 1 mmol) were dissolved in acetonitrile (10 mL) andtriethylamine (1 mL) and heated to 80° C. for 2 hours in a sealed tube.The reaction mixture was cooled to room temperature and evaporated todryness. The crude material was purified by reverse phase preparativeHPLC to afford the title compound (25 mg, 18% yield). ¹H NMR (400 MHz,DMSO): δ 8.07 (dd, J=2.8, 10.1 Hz, 1H), 7.91 (m, 2H), 7.74 (dd, J=5.6,5.6 Hz, 1H), 7.56-7.50 (m, 1H), 7.43 (dd, J=4.8, 9.1 Hz, 1H), 5.38 (s,1H), 4.98 (d, J=4.5 Hz, 1H), 4.78 (s, 2H), 4.14-4.06 (m, 1H), 3.98-3.93(m, 1H), 3.44-3.48 (m, 1H), 3.29-3.20 (m, 1H), 3.04 (dd, J=11.5, 25.1Hz, 2H), 2.67-2.58 (m, 1H), 2.50-2.38 (m, 2H), 2.25 (dd, J=11.7, 15.5Hz, 2H), 1.74-1.68 (m, 2H).

Preparation of Compound 314

Compound 314 was prepared as described herein below.

Step 1—Synthesis of 5-(benzyloxy)-2-(hydroxymethyl)pyridin-4(1H)-one

A suspension of 5-(benzyloxy)-2-(hydroxymethyl)-4H-pyran-4-one (53.2 g,229 mmol) in IMS (75 mL) and aq. NH₄OH solution (400 mL) was stirred at70° C. for 18 h. The cooled solution was diluted with water (400 mL),cooled to 5° C. and the suspension stirred for 30 min. The solid wasfiltered and dried under vacuum to leave the title compound as a palebrown solid (42.5 g, 80% yield). LC-MS (M−H⁺)=232. ¹H NMR (300 MHz,DMSO-d6): δ 11.08 (br s, 1H), 7.51-7.28 (m, 6H), 6.16 (br s, 1H), 5.56(br s, 1H), 5.01 (s, 2H), 4.34 (s, 2H).

Step 2—Synthesis of 5-(benzyloxy)-3-bromo-2-(hydroxymethyl)pyridin-4(1H)-one

A suspension of 5-(benzyloxy)-2-(hydroxymethyl)pyridine-4(1H)-one (11.6g, 50.0 mmol) and NBS (10.2 g, 57.5 mmol) in AcOH (75 mL) was stirred at40° C. for 1 h. The cooled suspension was filtered, the solid washedwith AcOH (25 mL) and dried under vacuum to leave the title compound asa pale yellow solid (13.7 g, 89% yield). LC-MS (M−H⁺)=310-312. ¹H NMR(300 MHz, DMSO-d6): δ 7.56 (s, 1H), 7.47-7.30 (m, 5H), 5.12 (s, 2H),4.56 (s, 2H).

Step 3—Synthesis of 5-bromo-6-(hydroxymethyl)pyridine-3,4-diolhydrochloride

A suspension of 5-(benzyloxy)-3-bromo-2-(hydroxymethyl)pyridin-4(1H)-one(3.07 g, 9.90 mmol) in water (10 mL) and conc. aq. HCl solution (10 mL)was stirred at 100° C. for 2 h. The mixture was cooled to 0° C., thenthe suspension filtered. The solid was washed with EtOAc (10 mL) thendried under vacuum to leave a brown solid (1.93 g, 76% yield). LC-MS(M−H⁺)=220-222. ¹H NMR (300 MHz, DMSO-d6): δ 7.88 (s, 1H), 4.63 (s, 2H),2.42 (s, 1H).

Step 4—Synthesis of(8-bromo-2,3-dihydro[1,4]dioxino[2,3-c]pyridin-7-yl)methanol

A suspension of 5-bromo-6-(hydroxymethyl)pyridine-3,4-diol hydrochloride(1.93 g, 7.52 mmol), 1,2-dibromoethane (2.12 g, 11.3 mmol) and K₂CO₃(1.56 g, 11.3 mmol) in DMF (5 mL) was stirred at 75° C. for 5 h. Thecooled suspension was concentrated under vacuum, then suspended inDCM-MeOH (98:2, 25 mL) and filtered through Celite. The filter cake waswashed with DCM-MeOH (98:2, 50 mL) then the combined organics wereconcentrated under vacuum to leave a pale brown crystalline solid (1.30g, 70% yield). LC-MS (M−H⁺)=246-248. ¹H NMR (300 MHz, DMSO-d6): δ 8.08(s, 1H), 4.66 (s, 2H), 4.50-4.46 (m, 2H), 4.35-4.30 (m, 2H).

Step 5—Synthesis of8-bromo-2,3-dihydro[1,4]dioxino[2,3-c]pyridine-7-carbaldehyde

A suspension of(8-bromo-2,3-dihydro-[1,4]dioxino[2,3-c]pyridin-7-yl)methanol (1.25 g,5.08 mmol) and MnO₂ (4.42 g, 51 mmol) in dioxane (25 mL) was stirred at90° C. for 5 h. The suspension was cooled to RT then was filteredthrough Celite, and the filter cake washed with warm dioxane (40° C., 25mL). The combined organics were concentrated under vacuum to leave apale orange solid (715 mg, 58% yield). ¹H NMR (300 MHz, CDCl3): δ 10.16(s, 1H), 8.29 (s, 1H), 4.54-4.49 (m, 2H), 4.43-4.39 (m, 2H).

Step 6—Synthesis of 9-tert-butyl 8-methyl2,3-dihydro-9H-[1,4]dioxino[2,3-d]pyrrolo[3,2-b]pyridine-8,9-dicarboxylate

To a solution of8-bromo-2,3-dihydro-[1,4]dioxino[2,3-c]pyridine-7-carbaldehyde (715 mg,2.93 mmol) and N-Boc-2-phosphonoglycine trimethyl ester (1.09 g, 3.66mmol) in DCM (20 mL) at RT was added DBU (558 mg, 3.66 mmol) and theresulting mixture stirred at RT for 30 min. Aq. citric acid solution(10%, 20 mL) was added, then the aq. layer extracted with DCM (20 mL).The combined organics were passed through a hydrophobic frit andconcentrated under vacuum to 3 mL volume. FCC (10-35% EtOAc iniso-hexane) gave the intermediate methyl(Z)-3-(8-bromo-2,3-dihydro-[1,4]dioxino[2,3-c]pyridin-7-yl)-2-((tert-butoxycarbonyl)-amino)acrylateas an off-white solid (835 mg, 69% yield, LC-MS (M−H⁺)=415-417). N₂ wasbubbled through a suspension of the intermediate, K₃PO₄ (862 mg, 4.06mmol) and 3 Å MS (400 mg) in t-BuOH (40 mL) at 50° C. for 30 min.BrettPhos Pd G3 (CAS: 1470372-59-8; 92 mg, 0.10 mmol) was added, thenthe flask evacuated and purged with N₂ thrice. The mixture was stirredat 85° C. for 4.5 h. The cooled suspension was diluted with EtOAc (40mL) then the mixture filtered through Celite. The filter cake was washedwith EtOAc (40 mL), then the combined organics concentrated under vacuumto leave a gum (810 mg). FCC (10-60% EtOAc in iso-hexane) gave acrystalline orange solid (543 mg, 80% yield). LC-MS (M−H⁺)=335. ¹H NMR(300 MHz, CDCl₃): δ 8.22 (s, 1H), 7.26 (s, 1H), 4.47-4.42 (m, 2H),4.41-4.36 (m, 2H), 3.93 (s, 3H), 1.64 (s, 9H).

Step 7—Synthesis of methyl2,3-dihydro-9H-[1,4]dioxino[2,3-d]pyrrolo[3,2-b]pyridine-8-carboxylate

A solution of 9-(tert-butyl) 8-methyl2,3-dihydro-9H-[1,4]dioxino[2,3-d]pyrrolo[3,2-b]pyridine-8,9-dicarboxylate(543 mg, 1.62 mmol) in TFA (2 mL) and DCM (10 mL) was stirred at refluxfor 1 h. The cooled solution was concentrated under vacuum, dissolved inMeOH (3 mL), applied to an SCX-2 cartridge (10 g) and washed with MeOH(40 mL). The product was eluted with a 2 M solution of NH₃ in MeOH (40mL); concentration under vacuum left the title product as a pale yellowsolid (346 mg, 91% yield). LC-MS (M−H⁺)=235. ¹H NMR (300 MHz, DMSO-d6):δ 12.22 (1H, br s), 8.08 (1H, s), 7.12 (1H, s), 4.50-4.44 (2H, m),4.41-4.35 (2H, m), 3.85 (3H, s).

Step 8—Synthesis of(2,3-dihydro-9H-[1,4]dioxino[2,3-d]pyrrolo[3,2-b]pyridin-8-yl)methanol

To a suspension of methyl2,3-dihydro-9H-[1,4]dioxino[2,3-d]pyrrolo[3,2-b]pyridine-8-carboxylate(344 mg, 1.47 mmol) in dry THF (20 mL) was added lithium aluminiumhydride (1 M in THF, 2.9 mL, 2.9 mmol) dropwise, at 0° C. under N₂. Thesuspension was stirred at 0° C. for 5 min and then at RT for 1 h. Thesuspension was cooled to 0° C., then water (0.12 mL), aq. NaOH solution(15%, 0.12 mmol), water (0.36 mL) and Na₂SO₄ were added sequentially.The suspension was stirred at RT for 30 min, then filtered throughCelite. The filter cake was washed with DCM-MeOH (9:1, 50 mL), then thecombined organics concentrated under vacuum to leave the title compoundas a pale yellow solid (110 mg, 36% yield). LC-MS (M−H⁺)=207. ¹H NMR(300 MHz, DMSO-d6): δ 11.18 (br s, 1H), 7.86 (s, 1H), 6.28 (s, 1H), 5.16(t, J=4.6 Hz, 1H), 4.55 (d, J=4.6 Hz, 2H), 4.47-4.42 (m, 2H), 4.34-4.29(m, 2H).

Step 9—Synthesis of2,3-dihydro-9H-[1,4]dioxino[2,3-d]pyrrolo[3,2-b]pyridine-8-carbaldehyde

A suspension of(2,3-dihydro-9H-[1,4]dioxino[2,3-d]pyrrolo[3,2-b]pyridin-8-yl)methanol(108 mg, 0.524 mmol) and MnO₂ (228 mg, 2.62 mmol) in dioxane (5 mL) wasstirred at 90° C. for 2 h. The suspension was cooled to 50° C., thenfiltered through Celite. The filter cake was washed with dioxane (10mL), then the combined organics concentrated under vacuum to leave thetitle compound as a yellow solid (84 mg, 79% yield). LC-MS (M−H⁺)=205.¹H NMR (300 MHz, DMSO-d6): δ 12.32 (br s, 1H), 9.83 (s, 1H), 8.13 (s,1H), 7.38 (s, 1H), 4.51-4.45 (m, 2H), 4.43-4.37 (m, 2H).

Step 10—Synthesis of1-[trans-4-{[(2,3-dihydro-9H-[1,4]dioxino[2,3-d]pyrrolo[3,2-b]pyridin-8-yl)methyl]amino}cyclohexyl]-7-fluoro-1H-pyrido[2,3-b][1,4]oxazin-2(3H)-one (compound 314)

A solution of1-(trans-4-aminocyclohexyl)-7-fluoro-1H-pyrido[2,3-b][1,4]oxazin-2(3H)-one(106 mg, 0.400 mmol),2,3-dihydro-9H-[1,4]dioxino[2,3-d]pyrrolo[3,2-b]pyridine-8-carbaldehyde(82 mg, 0.40 mmol), sodium triacetoxyborohydride (340 mg, 1.6 mmol) andAcOH (46 μL, 0.80 mmol) in MeCN (10 mL) was stirred at RT for 7 h. Themixture was concentrated under vacuum, suspended in a mixture of waterand sat. aq. NaHCO₃ solution (1:1, 10 mL) and extracted with DCM-MeOH(19:1, 2×10 mL). The combined organics were passed through a hydrophobicfrit and concentrated under vacuum to leave an orange-yellow gum. FCC(15 μm silica, 2-6% [2M NH₃ in MeOH] in DCM) followed by triturationwith warm MeCN (50° C.) provided the title compound as an off-whitesolid (89 mg, 49% yield). LC-MS (M−H⁺)=454.3. ¹H NMR (400 MHz, DMSO-d6):δ 11.09 (br s, 1H), 7.86-7.82 (m, 3H), 6.28 (s, 1H), 4.68 (s, 2H),4.48-4.44 (m, 2H), 4.34-4.31 (m, 2H), 4.01 (tt, J=3.6, 12.1 Hz, 1H),3.82 (s, 2H), 2.42 (tt, J=3.5, 11.0 Hz, 1H), 2.27 (dq, J=2.9, 12.6 Hz,2H), 2.14 (br s, 1H), 1.96 (apparent br d, J=11.9 Hz, 2H), 1.69(apparent br d, J=11.9 Hz, 2H), 1.24 (dq, J=3.4, 12.3 Hz, 2H).

Preparation of Compound 343

Compound 343 was prepared as described herein below.

Step 1—Synthesis of[(2S)-3,8-dioxo-1,2-dihydro-3H,8H-2a,5,8a-triazaacenaphthylen-2-yl]methylmethanesulfonate

To an ice-cooled suspension of(2S)-2-(hydroxymethyl)-1,2-dihydro-3H,8H-2a,5,8a-triazaacenaphthylene-3,8-dione(prepared as in WO2009/141398, 0.500 g, 2.28 mmol) in DCM (35 mL) wasadded Et₃N (0.48 mL, 3.4 mmol), then methanesulfonyl chloride (0.21 mL,2.7 mmol) was added over 3 min. The mixture was stirred at RT for 45 minthen purified directly by FCC (2-10% MeOH in DCM) to give the titlecompound (0.64 g, 94% yield). LC-MS (M−H⁺)=298. ¹H NMR (400 MHz,DMSO-d6): δ 7.88 (d, J=9.6 Hz, 1H), 7.79 (s, 1H), 6.27 (d, J=9.6 Hz,1H), 5.36-5.30 (m, 1H), 4.86 (dd, J=3.5, 11.2 Hz, 1H), 4.62 (dd, J=2.4,11.2 Hz, 1H), 4.43 (dd, J=10.1, 12.0 Hz, 1H), 4.25 (dd, J=5.1, 12.1 Hz,1H), 3.21 (s, 3H).

Step 2—Synthesis of 4-chloro-6-fluoro-2H-1-benzopyran-2-one

Et₃N (3.5 mL, 25 mmol) was added over 5 min to a suspension of6-fluoro-4-hydroxy-2H-1-benzopyran-2-one (3.0 g, 16.7 mmol) in POCl₃ (42mL, 450 mmol). The mixture was stirred at RT for 10 min then heated atreflux for 16 h. The reaction was cooled to RT and then concentratedunder reduced pressure. Toluene was added and evaporated. The residuewas partitioned between DCM and aq. sodium bicarbonate. The aqueousphase was extracted with DCM, then the combined organic extracts werewashed with water, dried over Na₂SO₄ and evaporated. The residue waspurified by FCC (2-6% EtOAc in toluene) to give the title compound(3.082 g, 93% yield). LC-MS (M−H⁺)=199. ¹H NMR (400 MHz, CDCl3): δ 7.57(dd, J=2.4, 8.2 Hz, 1H), 7.39-7.32 (m, 2H), 6.67 (s, 1H).

Step 3—Synthesis of tert-butyl4-{2-[(6-fluoro-2-oxo-2H-1-benzopyran-4-yl)amino]-1-hydroxyethyl}piperidine-1-carboxylate

Et₃N (1.0 mL, 7.2 mmol) was added to a solution of tert-butyl4-(2-amino-1-hydroxyethyl)piperidine-1-carboxylate (0.818 g, 3.34 mmol)in EtOH (25 mL). 4-chloro-6-fluoro-2H-1-benzopyran-2-one (0.65 g, 3.27mmol) was added and the mixture was stirred at RT for 5 min then heatedat 70° C. for 7.5 h. The reaction mixture was allowed to cool and thenevaporated. The residue was purified by FCC (2-8% [2M NH₃ in MeOH] inDCM) to give the title compound (0.859 g, 65% yield). LC-MS (M-Na⁺)=429.¹H NMR (400 MHz, DMSO-d6): δ 8.01 (dd, J=2.9, 10.0 Hz, 1H), 7.55 (br t,J=5.3 Hz, 1H), 7.50-7.45 (m, 1H), 7.37 (dd, J=4.9, 9.1 Hz, 1H), 5.23 (s,1H), 4.95 (d, J=5.3 Hz, 1H), 4.04-3.91 (m, 2H), 3.62-3.57 (m, 1H),3.36-3.30 (m, 1H, under water peak), 3.18-3.11 (m, 1H), 2.74-2.53 (m,2H), 1.77-1.69 (m, 1H), 1.61-1.53 (m, 2H), 1.39 (s, 9H), 1.32-1.13 (m,2H).

Step 4—Synthesis of6-fluoro-4-{[2-hydroxy-2-(piperidin-4-yl)ethyl]amino}-2H-1-benzopyran-2-one

TFA (7.5 mL) was added to an ice-cooled solution of tert-butyl4-{2-[(6-fluoro-2-oxo-2H-1-benzopyran-4-yl)amino]-1-hydroxyethyl}piperidine-1-carboxylate(0.859 g, 2.11 mmol) in DCM (30 mL). The mixture was stirred at RT for 1h. Toluene was added and evaporated. The residue was dissolved in MeOH(25 mL), applied to an SCX-2 cartridge and washed with MeOH. The productwas eluted with a 2 M solution of NH₃ in MeOH and the evaporation gavethe title compound (0.502 g, 78% yield). LC-MS (M−H⁺)=307. ¹H NMR (400MHz, DMSO-d6): δ 8.03 (dd, J=2.9, 10.1 Hz, 1H), 7.57 (t, J=5.4 Hz, 1H),7.50-7.45 (m, 1H), 7.37 (dd, J=4.8, 9.1 Hz, 1H), 5.20 (s, 1H), 4.88 (brs, 1H), 3.56-3.51 (m, 1H), 3.18-3.08 (m, 2H), 2.99-2.90 (m, 2H),2.46-2.37 (m, 2H), 1.70-1.65 (m, 1H), 1.55-1.41 (m, 2H), 1.31-1.13 (m,2H).

Step 5—Synthesis of(2R)-2-[(4-{2-[(6-fluoro-2-oxo-2H-1-benzopyran-4-yl)amino]-1-hydroxyethyl}piperidin-1-yl)methyl]-1,2-dihydro-3H,8H-2a,5,8a-triazaacenaphthylene-3,8-dione(compound 343, diastereomeric mixture)

A mixture of(S)-(3,8-dioxo-1,2-dihydro-3H,8H-2a,5,8a-triazaacenaphthylen-2-yl)methylmethane-sulfonate (0.300 g, 1.01 mmol),6-fluoro-4-{[2-hydroxy-2-(piperidin-4-yl)ethyl]amino}-2H-1-benzopyran-2-one(0.464 g, 1.51 mmol), pyridine (0.33 mL, 4.0 mmol) and i-PrOH (20 mL)was stirred in a sealed vial at 120° C. for 2.5 h. The cooled mixturewas evaporated to dryness and purified via FCC (2-14% [2M NH₃ in MeOH]in DCM) to give the title compound (0.167 g, 33% yield) as an off-whitesolid. LC-MS (M−H⁺)=508.4. ¹H NMR (400 MHz, DMSO-d6): δ 8.01 (dd, J=2.9,10.0 Hz, 1H), 7.85 (d, J=9.6 Hz, 1H), 7.73 (s, 1H), 7.52 (t, J=5.0 Hz,1H), 7.50-7.45 (m, 1H), 7.37 (dd, J=4.9, 9.1 Hz, 1H), 6.25 (d, J=9.5 Hz,1H), 5.21 (s, 1H), 5.08-5.02 (m, 1H), 4.88 (dd, J=1.0, 5.4 Hz, 1H), 4.33(dd, J=9.4, 11.8 Hz, 1H), 4.25 (dd, J=5.0, 11.7 Hz, 1H), 3.57-3.51 (m,1H), 3.32-3.28 (m, 1H), 3.15-3.08 (m, 1H), 3.03-2.95 (m, 2H), 2.78 (dd,J=8.9, 12.6 Hz, 1H), 2.71-2.65 (m, 1H), 2.18-2.00 (m, 2H), 1.75-1.66 (m,1H), 1.59-1.51 (m, 1H), 1.39-1.18 (m, 3H).

Preparation of Compound 354

Compound 354 was prepared as described herein below.

Step 1—Synthesis of tert-butyl(2-{[trans-4-(7-fluoro-2-oxo-2,3-dihydro-1H-pyrido[2,3-b][1,4]oxazin-1-yl)cyclohexyl]amino}ethyl)carbamate

A mixture oftrans-1-(4-aminocyclohexyl)-7-fluoro-1H-pyrido[2,3-b][1,4]oxazin-2(3H)-one(296 mg, 1.12 mmol), N-Boc-2-aminoacetaldehyde (178 mg, 1.12 mmol) and 3Å MS in MeOH (8 mL) was stirred at RT for 18 h. NaBH₄ (80 mg, 2.1 mmol)was added and the mixture stirred at RT for 24 h. The mixture wasfiltered through Celite, the filter cake washed with MeOH and DCM, thenthe combined organics concentrated under vacuum to leave a residue. FCC(0-5% [2N NH₃ in MeOH] in DCM) provided the title intermediate (180 mg,39% yield). LC-MS (M−H⁺)=409. ¹H NMR (400 MHz, CDCl3): δ 7.78 (d, J=2.6Hz, 1H), 7.19 (dd, J=2.5, 9.0 Hz, 1H), 4.89 (br s, 1H), 4.07 (tt, J=3.8,12.6 Hz, 1H), 3.22 (q, J=5.8 Hz, 2H), 2.77 (t, J=5.9 Hz, 2H), 2.57 (tt,J=3.7, 11.1 Hz, 1H), 2.41 (dq, J=3.3, 12.9 Hz, 2H), 2.10 (apparent br d,J=12.8 Hz, 2H), 1.83 (apparent br d, J=12.8 Hz, 2H), 1.51-1.45 (m, 11H),1.30-1.16 (m, 2H).

Step 2—Synthesis of1-{trans-4-[(2-aminoethyl)amino]cyclohexyl}-7-fluoro-1H-pyrido[2,3-b][1,4]oxazin-2(3H)-one

A mixture of tert-butyl(2-trans-4-(7-fluoro-2-oxo-2,3-dihydro-1H-pyrido[2,3-b][1,4]oxazin-1-yl)cyclohexyl)amino)ethyl)carbamate(180 mg, 0.44 mmol) in TFA (0.50 mL) and DCM (2 mL) was stirred at RTfor 1.5 h. The mixture was applied to an SCX-2 cartridge, washed withMeOH and then eluted with NH₃ in MeOH (2 N); concentration under vacuumleft a brown solid (113 mg, 83% yield). LC-MS (M−H⁺)=309. ¹H NMR (400MHz, CDCl3): δ 7.78 (d, J=2.6 Hz, 1H), 7.20 (dd, J=2.6, 9.0 Hz, 1H),4.67 (s, 2H), 4.06 (tt, J=3.9, 12.3 Hz, 1H), 2.83 (dd, J=5.3, 6.2 Hz,2H), 2.71 (dd, J=5.2, 6.4 Hz, 2H), 2.58 (tt, J=3.7, 11.1 Hz, 1H), 2.43(dq, J=3.4, 12.9 Hz, 2H), 2.15-2.09 (m, 2H), 1.87-1.81 (m, 2H), 1.25(ddt, J=3.6, 12.5, 12.3 Hz, 2H).

Step 3—Synthesis of7-fluoro-1-[trans-4-({2-[(6-fluoro-2-oxo-2H-1-benzopyran-4-yl)amino]ethyl}amino)cyclohexyl]-1H-pyrido[2,3-b][1,4]oxazin-2(3H)-one(compound 354)

To a solution of1-{trans-4-[(2-aminoethyl)amino]cyclohexyl}-7-fluoro-1H-pyrido[2,3-b][1,4]oxazin-2(3H)-one(110 mg, 0.36 mmol) and Et₃N (43 mg, 0.43 mmol) in MeCN (3 mL),6-fluoro-2-oxo-2H-chromen-4-yl trifluoromethanesulfonate was added andthe mixture stirred at RT for 2 h. The mixture was partitioned betweenwater and EtOAc, then the aq. phase extracted with EtOAc. The combinedorganics were dried (MgSO₄), filtered and concentrated under vacuum toleave the crude product. FCC (15 μm silica, 0-3.5% [2M NH₃ in MeOH] inDCM) gave an off-white solid. The solid was triturated with MeCN (1 mL)to leave a wet solid that was dried under vacuum at 60° C. to give thetitle compound (46 mg, 27% yield). LC-MS (M−H⁺)=471.3. ¹H NMR (400 MHz,DMSO-d6): δ 7.98 (dd, J=2.9, 10.0 Hz, 1H), 7.87-7.81 (m, 2H), 7.55 (s,1H), 7.48 (ddd, J=2.9, 8.0, 9.0 Hz, 1H), 7.37 (dd, J=4.8, 9.1 Hz, 1H),5.25 (s, 1H), 4.70 (s, 2H), 4.04 (tt, J=3.7, 11.9 Hz, 1H), 3.32 (2H,methylene signal under water peak), 2.82 (t, J=6.5 Hz, 2H), 2.50 (1H,methine signal under solvent peak), 2.35 (dq, J=2.9, 12.7 Hz, 2H), 1.94(d, J=11.9 Hz, 2H), 1.71 (d, J=11.4 Hz, 2H), 1.28-1.16 (m, 2H).

Biological Assays Example 1 Inhibition of DNA Gyrase and Topo IV in E.coli and S. aureus

The above compounds were tested for the inhibition of the enzyme DNAgyrase in a gyrase supercoiling assay and for the inhibition of theenzyme topoisomerase IV in a decatenation assay, in both Gram positiveand Gram negative bacteria, according to the following methods.

Both the assays were carried out according to a set-up method modifiedfrom the article to Blanche F, et al. “Differential Behaviors ofStaphylococcus aureus and Escherichia coli Type II DNA Topoisomerases”,Antimicrob. Agents Chemother., 1996, Vol. 40, No. 12 p. 2714-2720.

The compounds were screened at single concentration (200, 100 or 50 μM),in duplicate.

Ciprofloxacin and novobiocin were used as reference compounds, at singleconcentration of 200 and 50 μM, respectively.

DNA Gyrase Supercoiling Assay.

Reagents from S. aureus and E. coli Gyrase Supercoiling Assay kits(Inspiralis, UK) were used. A master mix with a total volume sufficientfor the number of reactions to perform was prepared with the followingreagents: 5× assay buffer, relaxed pBR322 substrate (0.5 μg/reaction),RNase-DNase free water. Aliquotes of this mix were dispensed in eachtube, then 10× compound stock solutions or vehicle control (DMSO), wereadded to each reaction tube.

Reaction was started with E. Coli (2 U/reaction) or S. aureus (1U/reaction) gyrase enzyme addition.

A sample added with an equal volume of dilution buffer was used asnegative control (without enzyme).

The reaction tubes were gentle vortexed and incubated 30 minutes at 37°C. Each reaction was stopped by adding 30 μl of Stop Buffer and 30 μlchloroform/isoamyl alcohol (24/1), briefly vortexed for 5-10 seconds andcentrifuged at 20000×g for 2 minutes. Samples were loaded onto 1%agarose gel and subjected to electrophoresis for 1 hour at 80V constantvoltage in TAE (40 mM Tris-acetate, 2 mM EDTA).

Data acquisition and analysis. Treatment of relaxed pBR322 with DNAgyrase converted the relaxed topoisomers (DNAs of different linkingnumber) to the supercoiled form of the plasmid, which migrates faster onan agarose gel. An upper band might also be visible, which consists ofopen-circular (nicked) DNA which is present in the relaxed substrate butco-migrates with some of the relaxed topoisomers.

Bands were visualized by ethidium bromide staining (dilution 1:20000)for 30 minutes followed by destaining in distilled water for 10 minutes.

In order to evaluate the compounds activity on the enzyme, the bands ofsupercoiled DNAs in the gel were photographed by a digital imagingsystem ImageQuant LAS 4000 (GE Healthcare) according to manufacturer'sinstructions.

The fluorescent intensity of each band was analyzed by ImageQuant TLsoftware and it was expressed as volume (volume of the uncalibratedquantity of material in the image feature after subtraction of thebackground intensity by using rolling ball method).

Each band intensity was compared, as percentage, to vehicle sample bandintensity, which served as positive control, on the same gel.

Inhibitory activity was expressed as percent of inhibition versus thepositive control.

The results are summarized in the following Table 2.

Topoisomerase IV Decatenation Assay

S. aureus and E. coli Topoisomerase IV decatenation kits (Inspiralis,UK) were used. A master mix with a total volume sufficient for thenumber of reactions to perform was prepared with the following reagents:5× assay buffer (50 mM HEPES-KOH (pH 7.6), 100 mM potassium glutamate,10 mM magnesium acetate, 10 mM DTT, 1 mM ATP, 50 μg/ml albumin), kDNAsubstrate (200 ng/reaction), RNase-DNase free water. Aliquots of thismix were dispensed in each tube, then 10× compound stock solutions orvehicle control (DMSO), were added in each reaction tube.

Reaction was started with Topoisomerase IV enzyme (0.5 U/reaction)addition.

A sample added with an equal volume of dilution buffer was used asnegative control (without enzyme).

The reaction tubes were gentle vortexed and incubated 30 minutes at 37°C. Each reaction was stopped by adding 30 μl of Stop Buffer and 30 μl ofchloroform/isoamyl alcohol (24/1), briefly vortexed for 5-10 seconds andcentrifuged at 20000×g for 2 minutes. Samples taken from the upper phasewere loaded into 1% agarose gel and subjected to electrophoresis for 1hour at 80V constant voltage in TAE (40 mM Tris-acetate, 2 mM EDTA).

Data acquisition and analysis. Due to the high molecular mass, kDNAcould not enter an agarose gel under normal electrophoresis conditions,but remained in the wells. In the presence of Topo IV topoisomerasemini-circles (2.5 Kb) were released from kDNA by decatenation and werequickly and easily resolved in the gel at relatively high voltages.

Bands were visualized by ethidium bromide staining (dil 1:20000) for 30minutes followed by destaining in distilled water for 10 minutes.

For single concentration screening assay, in order to evaluate thecompounds activity on the enzymes, the bands of decatenated DNAs in thegel were photographed by a digital imaging system ImageQuant LAS 4000(GE Healthcare) according to manufacturer's instructions.

The fluorescent intensity of each band was analyzed by ImageQuant TLsoftware and it was expressed as volume (volume of the uncalibratedquantity of material in the image feature after subtraction of thebackground intensity by using rolling ball method).

Each band intensity was compared, as percentage, to vehicle sample bandintensity, which served as positive control, on the same gel.

Inhibitory activity was expressed as percent of inhibition versus thepositive control.

The results are summarized in the following Table 2.

TABLE 2 E. coli S. aureus % % inhibition % inhibition % Compound conc.DNA inhibition DNA inhibition No. (μm) gyrase Topo IV gyrase Topo IV 15750 83 100 89 100 160 50 50 60 65 n/a 164 50 100 100 100 100 165 50 70 90100 100 180 50 58 57 90  50 181 50 53 91 92  50 182 50 61 52 92  50a 18350 81 77 84  85 193 50 77 50 86  74 194 50 79 50 60  50 197 50 87 63 90 79 200 50 90 100 100 100 201 50 100 100 100 100 202 50 100 100 100 100204 50 100 100 100 100 205 50 100 100 100 100 206 50 100 100 100 100 20750 100 100 100 100 208 50 100 100 100 100 209 50 100 100 100 100 210 5092 86 100  84 211 50 100 100 100 100 212 50 100 100 100 100 213 50 98 97100  84 214 50 99 100 100 100 215 50 69 n/a 96 n/a 216 50 100 100 100100 217 50 82 50 100 100 219 50 100 100 100 100 220 50 100 50 100 100221 50 81 50 100 100 n/a = not active

The above results showed that the exemplified compounds effectivelyinhibited both DNA gyrase and Topo IV of E. coli, which is a Grampositive bacterium, and/or S. aureus, which is a Gram negativebacterium.

Example 2 Determination of IC₅₀

The compounds that in the above example 1 showed an inhibitory activitywere further assayed in concentration-response curve (eight half-logconcentrations ranging from 0.1 to 300 μM) in order to determine theIC₅₀.

The supercoiled or decatenated DNA bands obtained as described inExample 1 were analysed as follows.

Bands were analyzed by gel documentation equipment (Syngene, Cambridge,UK) and quantitated using Syngene Gene Tools software. Raw gel data(fluorescent band volumes) collected from Syngene, GeneTools gelanalysis software were converted to a percentage of the 100% control(the fully supercoiled or decatenated DNA band). These data wereanalyzed using SigmaPlot Version 12.3 (2013). The IC50 data werecalculated by using the global curve fit non-linear regression tool byselecting the Single, 2 Parameter fit function from the ExponentialDecay equation category.

The results are reported in the following table 3.

TABLE 3 E. coli S. aureus Compound IC₅₀ IC₅₀ IC₅₀ IC₅₀ No. DNA gyraseTopo IV DNA gyrase Topo IV 157 2.34 0.64 2.48 1.02 160 n/a 21.5 20.8 n/a164 3.77 1.78 1.46 0.7 165 5.25 1.24 3.60 1.12 180 0.48 3.78 1.70 0.11181 0.44 3.67 0.73 0.36 182 0.74 3.21 0.64 0.74 183 0.77 2.48 0.74 0.19193 0.25 1.38 0.28 2.25 194 0.25 6.06 0.49 0.69 200 0.04 <0.1 <0.1 <0.1201 <0.1 <0.1 0.19 <0.1 204 0.05 <0.1 0.64 <0.1 205 0.1 0.31 0.17 0.1206 0.05 <0.1 0.13 <0.1 207 <0.1 <0.1 <0.1 <0.1 208 0.08 0.27 0.11 0.13209 0.13 1.00 0.3 0.22 210 0.44 13.77 1.3 1.31 211 <0.1 <0.1 <0.1 <0.1212 0.1 0.17 0.14 0.07 213 0.11 1.00 0.28 0.58 214 0.12 1.03 0.16 0.57216 0.13 0.25 0.31 0.29 217 1.45 0.69 2.15 2.72 219 0.22 0.22 0.22 0.65220 0.68 0.74 1.97 2.91 221 0.92 0.67 2.76 2.63

Example 3 Determination of IC₅₀

Compounds 301-303, 314, 343 and 354 were assayed inconcentration-response curve (eight half-log concentrations ranging from0.1 to 300 μM) in order to determine the IC₅₀, following the proceduredescribed in example 2.

The results are reported in the following table 4.

TABLE 4 E. coli S. aureus Compound IC₅₀ IC₅₀ IC₅₀ IC₅₀ No. DNA gyraseTopo IV DNA gyrase Topo IV 301 0.46 0.43 0.72 70.3 302 0.59 0.12 0.583.26 303 2.67 0.62 9.92 >100 314 0.85 1.14 7.1 >100 343 0.91 1.27 1.988.73 354 0.47 0.18 0.35 0.92

The data of Table 3 are comparable to the data of Table 2, and confirmedthe activity of Compounds 301-303, 314, 343 and 354 to effectivelyinhibit both DNA gyrase and Topo IV of E. coli and/or S. aureus.

1: A compound of formula (1):A-L₁-Y-L₂-R—B  (1) wherein A is a cyclic group having the formula (II):

wherein G₃ is CH or N when the dashed line represents a double bond, orCH₂, NH or O when the dashed line represents a single bond; C₁represents the atoms necessary to form an aliphatic or aromaticsix-membered cycle optionally comprising one or more heteroatom selectedfrom nitrogen atom and oxygen atom, said cycle being optionallysubstituted by one or more substituent selected from the groupconsisting of halogen atom, (C₁₋₃)alkyl group, cyano group, oxo group(═O), and (C₁₋₃)alkoxy group; L₁ is σ bond, or (C₁₋₃)alkylenyl group,optionally substituted with hydroxy group; Y is a ring selected from thegroup consisting of piperidinyl, piperazinyl, pyrrolidinyl, 1-3cyclobutyl, 1-3 cyclopentyl, 1-2 cyclopropyl, azetidinyl,azabicyclo-octyl, morpholinyl and cyclohexyl ring, said ring beingoptionally substituted by one or more substituent selected from thegroup consisting of hydroxy group, (C₁₋₃)alkylenyl-OH group,(C₁₋₃)alkylenyl-O—(C₁₋₃)alkyl group, (C₁₋₃)alkylenyl-CONR′R″ group, andCONR′R″ group, wherein R′ and R″ are hydrogen atom or (C₁₋₃)alkyl; L₂ isσ bond, —(C₁₋₃)alkylenyl- group, NR′″ group, NR′″—(C₁₋₃)alkylenyl group,(C₁₋₃)alkylenyl-NR′″ group, NR′″—(C₁₋₃)alkylenyl-NR′″ group, or(C₁₋₃)alkylenyl-NR′″—(C₁₋₃)alkylenyl group, said group being optionallysubstituted with a hydroxy group, wherein R′″ is hydrogen, (C₁₋₃)alkyl,(C₁₋₃)alkylenyl-OH, (C₁₋₃)alkylenyl-O—(C₁₋₃)alkyl, or(C₁₋₃)alkylenyl-CONR′R″, wherein R′ and R″ are hydrogen atom or(C₁₋₃)alkyl; R is σ bond or heterocyclic ring, aliphatic or aromatic,having 5 members containing one or more nitrogen atoms, optionallysubstituted with CH₂OH, CH₂CN, CN or CONR′R″, wherein R′ and R″ arehydrogen atom or (C₁₋₃)alkyl; and B is cyclic group having the followingformula (VI):

wherein G₆ is CH or N; and R₂ is hydrogen atom or halogen atom; or anaddition salt with a pharmaceutically acceptable organic or inorganicacid or base, an enantiomer, an oxide thereof, or a quaternary ammoniumsalt thereof. 2: The compound according to claim 1, wherein R₂ isselected from the group consisting of a hydrogen atom, a fluorine atom,a chlorine atom, and a bromine atom. 3: The compound according to claim1, wherein R₂ is selected from the group consisting of a hydrogen atomand a fluorine atom. 4: The compound according to claim 1, wherein L₁ isa σ bond or a methylene group (—CH₂—). 5: The compound according toclaim 1, wherein L₂ is a σ bond, a (C₁₋₃)alkylenyl group, NR′″ group,—NR′″—(C₁₋₃)alkylenyl group, (C₁₋₃)alkylenyl-NR′″— group,—NR′″—(C₁₋₃)alkylenyl-NR′″— group, or(C₁₋₃)alkylenyl-NR′″—(C₁₋₃)alkylenyl group, said group being optionallysubstituted with one or more hydroxy group. 6: The compound according toclaim 5, wherein L₂ is a σ bond, a (C₁₋₂)alkylenyl group, NR′″ group,—NR′″—(C₁₋₂)alkylenyl group, (C₁₋₂)alkylenyl-NR′″— group,—NR′″—(C₁₋₂)alkylenyl-NR′″— group, or(C₁₋₂)alkylenyl-NR′″—(C₁₋₂)alkylenyl group, said group being optionallysubstituted with one hydroxy group. 7: The compound according to claim1, wherein R is a σ bond or an aromatic heterocyclic ring having 5members containing one or more nitrogen atoms, optionally substitutedwith CH₂CN or CN. 8: The compound according to claim 7, wherein R is a 6bond, a 1H-imidazol-4-yl group, or a 1H-pyrrol-2-yl group, optionallysubstituted with CH₂CN or CN 9: The compound according to claim 1,wherein A is a cyclic group having one of the following formulae:

10: The compound according to claim 1, wherein Y is a ring having one ofthe following formulae:

11: The compound according to claim 1, wherein B is a cyclic grouphaving one of the following formulae:

12: A pharmaceutical composition, comprising at least one compound, saltthereof with a pharmaceutically acceptable organic or inorganic acid orwith a pharmaceutically acceptable organic or inorganic base,enantiomer, quaternary ammonium salt thereof, or N-oxide thereofaccording to claim 1, and at least one inert pharmaceutically acceptableexcipient. 13: The compound according to claim 1, for use in medicine.14: The compound according to claim 1, for use in the treatment of abacterial infection. 15: The compound according to claim 14, whereinsaid bacterial infection is selected from the group consisting of a skininfection, a mucosal infection, a gynecological infection, a respiratorytract infection (RTI), a CNS infections, a gastro-intestinal infection,a bone infection, a cardiovascular infection, a sexually transmittedinfection, and a urinary tract infection. 16: A method for treating abacterial infection, comprising administering an effective amount of acompound, salt thereof with a pharmaceutically acceptable organic orinorganic acid or with a pharmaceutically acceptable organic orinorganic base, enantiomer, quaternary ammonium salt thereof, or N-oxidethereof according to claim 1 to a patient in need thereof. 17: Themethod according to claim 16, wherein said bacterial infection isselected from the group consisting of a skin infection, a mucosalinfection, a gynecological infection, a respiratory tract infection(RTI), a CNS infections, a gastro-intestinal infection, a boneinfection, a cardiovascular infection, a sexually transmitted infection,and a urinary tract infection.